U.S. patent application number 11/686850 was filed with the patent office on 2007-10-18 for polyunsaturated fatty acid production in heterologous organisms using pufa polyketide synthase systems.
This patent application is currently assigned to MARTEK BIOSCIENCES CORPORATION. Invention is credited to Jerry M. Kuner, James Casey Lippmeier, James G. Metz, Maurice Martin Moloney, Cory Lee Nykiforuk.
Application Number | 20070245431 11/686850 |
Document ID | / |
Family ID | 38510298 |
Filed Date | 2007-10-18 |
United States Patent
Application |
20070245431 |
Kind Code |
A1 |
Metz; James G. ; et
al. |
October 18, 2007 |
POLYUNSATURATED FATTY ACID PRODUCTION IN HETEROLOGOUS ORGANISMS
USING PUFA POLYKETIDE SYNTHASE SYSTEMS
Abstract
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.
Inventors: |
Metz; James G.; (Longmont,
CO) ; Kuner; Jerry M.; (Longmont, CO) ;
Lippmeier; James Casey; (Columbia, MD) ; Moloney;
Maurice Martin; (Calgary, CA) ; Nykiforuk; Cory
Lee; (Calgary, CA) |
Correspondence
Address: |
SHERIDAN ROSS PC
1560 BROADWAY
SUITE 1200
DENVER
CO
80202
US
|
Assignee: |
MARTEK BIOSCIENCES
CORPORATION
6480 Dobbin Road
Columbia
MD
21045
SEMBIOSYS GENETICS INC.
110, 2985-23rd Avenue N.E.
Calgary
T1Y 7L3
|
Family ID: |
38510298 |
Appl. No.: |
11/686850 |
Filed: |
March 15, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60783205 |
Mar 15, 2006 |
|
|
|
60784616 |
Mar 21, 2006 |
|
|
|
Current U.S.
Class: |
800/281 ;
435/190; 435/419; 435/468 |
Current CPC
Class: |
A61P 1/16 20180101; C12N
15/8247 20130101; A61P 11/00 20180101; C12P 7/6427 20130101; Y02P
20/52 20151101; A61P 19/02 20180101; A61P 25/24 20180101; A61P 9/10
20180101; A61P 35/00 20180101; C12N 9/93 20130101; C12N 9/1029
20130101; A61P 7/00 20180101; A61P 15/00 20180101; A61P 3/06
20180101; A61P 31/04 20180101; C12P 7/6472 20130101; A61P 19/10
20180101; A61P 25/08 20180101; A61P 25/28 20180101; A61P 15/06
20180101; A61P 37/06 20180101; A61P 43/00 20180101; A61P 1/04
20180101; A61P 19/08 20180101; A61P 29/00 20180101; A61P 3/02
20180101; A61P 27/02 20180101 |
Class at
Publication: |
800/281 ;
435/419; 435/468; 435/190 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C12N 9/04 20060101 C12N009/04; C12N 15/82 20060101
C12N015/82; C12N 5/04 20060101 C12N005/04 |
Claims
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 .beta.-ketoacyl-ACP synthase
(KS) domains; d) at least one acyltransferase (AT) domain; e) at
least one .beta.-ketoacyl-ACP reductase (KR) domain; f) at least
two FabA-like .beta.-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 .beta.-keto acyl-ACP synthase (KS) domains;
d) one acyltransferase (AT) domain; e) one ketoreductase (KR)
domain; f) two FabA-like .beta.-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.degree. 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 .beta.-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
.beta.-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.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) from U.S. Provisional Application Ser. No.
60/784,616, filed Mar. 21, 2006, and from U.S. Provisional
Application Ser. No. 60/783,205, filed Mar. 15, 2006. The entire
disclosure of each of U.S. Provisional Application Ser. No.
60/784,616 and U.S. Provisional Application Ser. No. 60/783,205,
filed Mar. 15, 2006 is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention generally relates to the use of
accessory proteins and targets to improve the production of
polyunsaturated fatty acids (PUFAs) and particularly, long chain
PUFAs (LCPUFAs), in a host organism that has been genetically
modified with a PKS-like system for producing such PUFAs (i.e., a
PUFA PKS system or a PUFA synthase). The present invention also
relates to the organisms that have been genetically modified to
express such accessory proteins or modified with respect to such
targets, and to methods of making and using such organisms.
BACKGROUND OF THE INVENTION
[0003] Polyunsaturated fatty acids (PUFAs) are considered to be
useful for nutritional applications, pharmaceutical applications,
industrial applications, and other purposes. However, the current
supply of PUFAs from natural sources and from chemical synthesis is
not sufficient for commercial needs. Vegetable oils derived from
oil seed crops are relatively inexpensive and do not have the
contamination issues associated with fish oils. However, the PUFAs
found in commercially developed plant oils are typically limited to
linoleic acid (eighteen carbons with 2 double bonds, in the delta 9
and 12 positions--18:2 delta 9,12) and linolenic acid (18:3 delta
9,12,15). A number of separate desaturase and elongase enzymes are
required for fatty acid synthesis from linoleic and linolenic acids
to produce the more saturated and longer chain PUFAs. Therefore,
engineering plant host cells for the expression of PUFAs such as
EPA and docosahexaenoic acid (DHA) may require expression of
several separate enzymes to achieve synthesis. Additionally, for
production of useable quantities of such PUFAs, additional
engineering efforts may be required. Therefore, the discovery of an
alternate system for the production of PUFAs, which is a polyketide
synthase-like system, has provided a significant alternative to the
genetic engineering of plants or other organisms (e.g.,
microorganisms) using the desaturases and elongases of the
"classical" or "standard" fatty acid synthesis pathway.
[0004] There have been many efforts to produce PUFAs in oil-seed
crop plants by modification of the endogenously-produced fatty
acids. Genetic modification of these plants with various individual
genes for fatty acid elongases and desaturases has produced leaves
or seeds containing significant levels of PUFAs such as EPA, but
also containing significant levels of mixed shorter-chain and less
unsaturated PUFAs (Qi et al., Nature Biotech. 22:739 (2004); PCT
Publication No. WO 04/071467; Abbadi et al., Plant Cell 16:1
(2004)); Napier and Sayanova, Proceedings of the Nutrition Society
(2005), 64:387-393; Robert et al., Functional Plant Biology (2005)
32:473-479; or U.S. Patent Application Publication
2004/0172682.
[0005] Therefore, there remains a need in the art for a method to
efficiently and effectively produce quantities of lipids (e.g.,
triacylglycerol (TAG) and phospholipid (PL)) enriched in desired
PUFAs in oil-seed plants.
[0006] Polyketide synthase (PKS) systems are generally known in the
art as enzyme complexes related to fatty acid synthase (FAS)
systems, but which are often highly modified to produce specialized
products that typically show little resemblance to fatty acids. It
has now been shown, however, that polyketide synthase systems exist
in marine bacteria and certain microalgae that are capable of
synthesizing polyunsaturated fatty acids (PUFAs) from acetyl-CoA
and malonyl-CoA. These systems are referred to herein as PUFA PKS
systems, PKS-like systems for the production of PUFAs, or PUFA
synthase systems, all of which are used interchangeably herein.
[0007] The PUFA PKS pathways for PUFA synthesis in Shewanella and
another marine bacteria, Vibrio marinus, are described in detail in
U.S. Pat. No. 6,140,486. The PUFA PKS pathways for PUFA synthesis
in the eukaryotic Thraustochytrid, Schizochytrium, is described in
detail in U.S. Pat. No. 6,566,583. The PUFA PKS pathways for PUFA
synthesis in eukaryotes such as members of Thraustochytriales,
including the additional description of a PUFA PKS system in
Schizochytrium and the identification of a PUFA PKS system in
Thraustochytrium, including details regarding uses of these
systems, are described in detail in U.S. Patent Application
Publication No. 20020194641, published Dec. 19, 2002 and in PCT
Publication No. WO 2006/135866, published Dec. 21, 2006. U.S.
Patent Application Publication No. 20040235127, published Nov. 25,
2004, discloses the detailed structural description of a PUFA PKS
system in Thraustochytrium, and further detail regarding the
production of eicosapentaenoic acid (C20:5, .omega.-3) (EPA) and
other PUFAs using such systems. U.S. Patent Application Publication
No. 20050100995, published May 12, 2005, discloses the structural
and functional description of PUFA PKS systems in Shewanella
olleyana and Shewanella japonica, and uses of such systems. These
applications also disclose the genetic modification of organisms,
including microorganisms and plants, with the genes comprising the
PUFA PKS pathway and the production of PUFAs by such organisms.
Furthermore, PCT Patent Publication No. WO 05/097982 describes a
PUFA PKS system in Ulkenia, and U.S. Patent Application Publication
No. 20050014231 describes PUFA PKS genes and proteins from
Thraustochytrium aureum. Each of the above-identified applications
is incorporated by reference herein in its entirety.
[0008] Accordingly, the basic domain structures and sequence
characteristics of the PUFA synthase family of enzymes have been
described, and it has been demonstrated that PUFA synthase enzymes
are capable of de novo synthesis of various PUFAs (e.g.,
eicosapentaenoic acid (EPA; C20:5n-3), docosahexaenoic acid (DHA;
22:6n-3) and docosapentaenoic acid (DPAn-6; C22:5n-6). It has also
been demonstrated that the PUFA products can accumulate in host
organism phospholipids (PL) and, in some cases, in the neutral
lipids (e.g., triacylglycerols (TAG)). However, to the best of the
present inventors' knowledge, the precise mechanism for the
transfer of the PUFA from the enzyme to those targets has not been
defined prior to the present invention.
[0009] Since the mechanism of transfer of PUFAs to target
destinations in an organism can have implications for increasing
the efficiency of and/or improving the production of PUFAs in an
organism that has been genetically modified to produce such PUFAs,
there is a need in the art for information regarding this
mechanism. Accordingly, there is also a need in the art for
improved methods of production of PUFAs, including in plants and
microorganisms that have been genetically modified to produce such
PUFAs, which take advantage of the knowledge of such mechanism.
SUMMARY OF THE INVENTION
[0010] One embodiment of the invention relates to an isolated
nucleic acid molecule comprising 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, wherein the
nucleic acid sequence encodes 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. In one aspect, the nucleic
acid sequence encodes 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. In one
aspect, the nucleic acid sequence encodes an amino acid sequence
selected from the group consisting of: SEQ ID NO:83, SEQ ID NO:85
and SEQ ID NO:97. In one aspect, the nucleic acid sequence is
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.
[0011] Yet another embodiment of the invention relates to an
isolated nucleic acid molecule comprising a nucleic acid sequence
that encodes a protein that utilizes PUFA-CoA as substrates in
forming phospholipids (PL) or triacylglycerols (TAG), wherein the
protein comprises 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:110, and SEQ ID NO:113. In one aspect, the nucleic acid sequence
encodes 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:100, and SEQ ID NO:113. In one aspect, the
nucleic acid sequence encodes a protein comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:102 and
SEQ ID NO:104. In one aspect, the nucleic acid sequence is 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.
[0012] Another embodiment of the invention relates to an isolated
protein encoded by any of the above-described nucleic acid
molecules.
[0013] Another embodiment of the invention relates to a recombinant
nucleic acid molecule, comprising any of the above-described
nucleic acid molecules, operatively linked to an expression control
sequence.
[0014] Yet another embodiment of the invention relates to a
recombinant host cell comprising any of the above-described
recombinant nucleic acid molecules. In one aspect, the host cell is
a microorganism. In another aspect, the host cell is a plant
cell.
[0015] Another embodiment of the invention relates to a genetically
modified organism, wherein the organism has been genetically
modified to express any of the above-described nucleic acid
molecules or any combination thereof. In one aspect, the organism
expresses a PUFA synthase and a phosphopantetheinyl transferase
(PPTase). In one aspect, the organism has been genetically modified
to express the synthase and the PPTase. In one aspect, the contains
an additional genetic modification to delete or inactivate a fatty
acid synthase (FAS) expressed by the organism. In one aspect, the
organism contains an additional genetic modification to reduce
competition for malonyl CoA with the PUFA synthase or to increase
the level of malonyl CoA in the organism.
[0016] Another embodiment relates to a genetically modified
organism, wherein the organism expresses a PUFA synthase that
produces at least one polyunsaturated fatty acid (PUFA) and a
phosphopantetheinyl transferase (PPTase), and wherein the organism
contains a 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. In one aspect, the organism is transformed with a nucleic
acid molecule comprising a nucleic acid sequence encoding an
acyl-CoA synthetase (ACoAS) or a homologue thereof from an organism
that endogenously expresses a PUFA synthase. In one aspect, the
organism 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. In one aspect, the organism is
transformed with a nucleic acid molecule comprising a nucleic acid
sequence encoding an acyl-CoA synthetase (ACoAS) or a homologue
thereof from a Thraustochytriales microorganism, wherein the ACoAS
or homologue thereof catalyzes the conversion of long chain PUFA
free fatty acids (FFA) to acyl-CoA. In one aspect, the organism is
transformed with a nucleic acid molecule comprising a nucleic acid
sequence encoding an acyl-CoA synthetase (ACoAS) or a homologue
thereof from Schizochytrium, wherein the ACoAS or homologue thereof
catalyzes the conversion of long chain PUFA free fatty acids (FFA)
to acyl-CoA. In one aspect, the organism contains an additional
genetic modification to delete or inactivate a fatty acid synthase
(FAS) expressed by the organism. In one aspect, the organism
contains an additional genetic modification to reduce competition
for malonyl CoA with the PUFA synthase or to increase the level of
malonyl CoA in the organism. In one aspect, the organism 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).
[0017] Another embodiment relates to a genetically modified
organism, wherein the organism expresses a PUFA synthase that
produces at least one polyunsaturated fatty acid (PUFA) and a
phosphopantetheinyl transferase (PPTase), and wherein the organism
contains a genetic modification to delete or inactivate a fatty
acid synthase (FAS) expressed by the organism. In one aspect, the
organism contains an additional genetic modification to reduce
competition for malonyl CoA with the PUFA synthase or to increase
the level of malonyl CoA in the organism.
[0018] Another embodiment relates to a genetically modified
organism, wherein the organism expresses a PUFA synthase that
produces at least one polyunsaturated fatty acid (PUFA) and a
phosphopantetheinyl transferase (PPTase), and wherein the organism
contains a genetic modification to reduce competition for malonyl
CoA with the PUFA synthase or to increase the level of malonyl CoA
in the organism. In one aspect, the organism contains an additional
genetic modification to delete or inactivate a fatty acid synthase
(FAS) expressed by the organism.
[0019] Yet another embodiment relates to a genetically modified
organism, wherein the organism expresses a PUFA synthase that
produces at least one polyunsaturated fatty acid (PUFA) and a
phosphopantetheinyl transferase (PPTase), wherein the organism
contains a 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). In one aspect, the
protein is a DAGAT or an LPAAT. In one aspect, the organism 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). In one aspect, the
organism 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). In one aspect, the organism comprises an
additional 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. In one aspect, the organism contains an additional
genetic modification to delete or inactivate a fatty acid synthase
(FAS) expressed by the organism. In one aspect, the organism
contains an additional genetic modification to reduce competition
for malonyl CoA with the PUFA synthase or to increase the level of
malonyl CoA in the organism.
[0020] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 organism contains a genetic
modification to inhibit the expression or activity of a protein
selected from the group consisting of KASII and KASIII.
[0021] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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.
[0022] In a further embodiment, the invention provides a
genetically modified organism, including a microorganism, plant,
part of the plant, or plant cell wherein the organism 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 organism contains a genetic
modification to inhibit the expression or activity of a protein
selected from the group consisting of KASII and KASIII, and wherein
the organism 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.
[0023] In yet a further embodiment, the invention provides a
genetically modified organism, including a microorganism, plant,
part of the plant, or plant cell wherein the organism has been
genetically modified with a PUFA synthase that produces at least
one polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); 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,
including, but not limited to that represented by SEQ ID NO:81; or
wherein the organism 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.
[0024] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism has been genetically
modified with a PUFA synthase that produces at least one
polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); wherein the organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII; wherein the organism contains an additional genetic
modification to express one or more heterologous proteins from an
organism that endogenously produces PUFAs; and wherein the protein
utilizes PUFA-CoA as substrates in forming phospholipids (PL) or
triacylglycerols (TAG).
[0025] In yet another embodiment, the invention provides a
genetically modified organism, including a microorganism, plant,
part of the plant, or plant cell, wherein the organism has been
genetically modified with a PUFA synthase that produces at least
one polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); wherein the organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII; wherein the organism 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); and wherein the organism 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.
[0026] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism has been genetically
modified with a PUFA synthase that produces at least one
polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); 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,
including, but not limited to that represented by SEQ ID NO:81;
wherein the organism 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).
[0027] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism has been genetically
modified with a PUFA synthase that produces at least one
polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); 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,
including, but not limited to that represented by SEQ ID NO:81;
wherein the organism 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); and wherein the organism 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.
[0028] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism has been genetically
modified with a PUFA synthase that produces at least one
polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); wherein the organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII; and wherein the organism comprises an additional genetic
modification to delete or inactivate an endogenous fatty acid
synthase (FAS) or protein associated with an FAS expressed by the
organism.
[0029] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism has been genetically
modified with a PUFA synthase that produces at least one
polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); wherein the organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII; wherein the organism comprises an additional genetic
modification to delete or inactivate an endogenous fatty acid
synthase (FAS) or protein associated with an FAS expressed by the
organism; and wherein the organism 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.
[0030] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism has been genetically
modified with a PUFA synthase that produces at least one
polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); wherein the organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII; wherein the organism comprises an additional genetic
modification to delete or inactivate an endogenous fatty acid
synthase (FAS) or protein associated with an FAS expressed by the
organism; wherein the organism contains an additional genetic
modification to express one or more heterologous proteins from an
organism that endogenously produces PUFAs; and wherein the protein
utilizes PUFA-CoA as substrates in forming phospholipids (PL) or
triacylglycerols (TAG).
[0031] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism has been genetically
modified with a PUFA synthase that produces at least one
polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); wherein the organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII; wherein the organism comprises an additional genetic
modification to delete or inactivate an endogenous fatty acid
synthase (FAS) or protein associated with an FAS expressed by the
organism; wherein the organism 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;
wherein the organism contains an additional genetic modification to
express one or more heterologous proteins from an organism that
endogenously produces PUFAs; and wherein the protein utilizes
PUFA-CoA as substrates in forming phospholipids (PL) or
triacylglycerols (TAG). In another embodiment, the invention
provides a genetically modified organism, including a
microorganism, plant, part of the plant, or plant cell, wherein the
organism has been genetically modified with a PUFA synthase that
produces at least one polyunsaturated fatty acid (PUFA) and a
phosphopantetheinyl transferase (PPTase); 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, including, but not limited to that
represented by SEQ ID NO:81; and wherein the organism comprises an
additional genetic modification to delete or inactivate an
endogenous fatty acid synthase (FAS) or protein associated with an
FAS expressed by the organism.
[0032] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism has been genetically
modified with a PUFA synthase that produces at least one
polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); 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,
including, but not limited to that represented by SEQ ID NO:81;
wherein the organism comprises an additional genetic modification
to delete or inactivate an endogenous fatty acid synthase (FAS) or
protein associated with an FAS expressed by the organism; and
wherein the organism 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.
[0033] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism has been genetically
modified with a PUFA synthase that produces at least one
polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); 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,
including, but not limited to that represented by SEQ ID NO:81;
wherein the organism comprises an additional genetic modification
to delete or inactivate an endogenous fatty acid synthase (FAS) or
protein associated with an FAS expressed by the organism; wherein
the organism contains an additional genetic modification to express
one or more heterologous proteins from an organism that
endogenously produces PUFAs; and wherein the protein utilizes
PUFA-CoA as substrates in forming phospholipids (PL) or
triacylglycerols (TAG).
[0034] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism has been genetically
modified with a PUFA synthase that produces at least one
polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl
transferase (PPTase); 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,
including, but not limited to that represented by SEQ ID NO:81;
wherein the organism comprises an additional genetic modification
to delete or inactivate an endogenous fatty acid synthase (FAS) or
protein associated with an FAS expressed by the organism; wherein
the organism 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; and wherein the organism
contains an additional genetic modification to express one or more
heterologous proteins from an organism that endogenously produces
PUFAs; and wherein the protein utilizes PUFA-CoA as substrates in
forming phospholipids (PL) or triacylglycerols (TAG).
[0035] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII, wherein the organism 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
organism.
[0036] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII, wherein the organism 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 organism;
and wherein the organism 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.
[0037] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII, wherein the organism 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 organism;
wherein the organism contains an additional genetic modification to
express one or more heterologous proteins from an organism that
endogenously produces PUFAs; and wherein the protein utilizes
PUFA-CoA as substrates in forming phospholipids (PL) or
triacylglycerols (TAG).
[0038] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII, wherein the organism 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 organism;
and wherein the organism comprises an additional genetic
modification to delete or inactivate an endogenous fatty acid
synthase (FAS) or protein associated with an FAS expressed by the
organism.
[0039] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII, wherein the organism 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 organism;
wherein the organism 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; wherein the organism contains
an additional genetic modification to express one or more
heterologous proteins from an organism that endogenously produces
PUFAs; and wherein the protein utilizes PUFA-CoA as substrates in
forming phospholipids (PL) or triacylglycerols (TAG). In one
embodiment, the invention provides a genetically modified organism,
including a microorganism, plant, part of the plant, or plant cell,
wherein the organism 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 organism contains a genetic modification to inhibit the
expression or activity of a protein, e.g., a protein selected from
the group consisting of KASII and KASIII, wherein the organism
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 organism; wherein the organism 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; and wherein the organism comprises an additional genetic
modification to delete or inactivate an endogenous fatty acid
synthase (FAS) or protein associated with an FAS expressed by the
organism.
[0040] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII, wherein the organism 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 organism;
wherein the organism contains an additional genetic modification to
express one or more heterologous proteins from an organism that
endogenously produces PUFAs; and wherein the protein utilizes
PUFA-CoA as substrates in forming phospholipids (PL) or
triacylglycerols (TAG); and wherein the organism comprises an
additional genetic modification to delete or inactivate an
endogenous fatty acid synthase (FAS) or protein associated with an
FAS expressed by the organism.
[0041] In one embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 organism contains a genetic
modification to inhibit the expression or activity of a protein,
e.g., a protein selected from the group consisting of KASII and
KASIII, wherein the organism 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 organism;
wherein the organism 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; wherein the organism contains
an additional genetic modification to express one or more
heterologous proteins from an organism that endogenously produces
PUFAs; and wherein the protein utilizes PUFA-CoA as substrates in
forming phospholipids (PL) or triacylglycerols (TAG); and wherein
the organism comprises an additional genetic modification to delete
or inactivate an endogenous fatty acid synthase (FAS) or protein
associated with an FAS expressed by the organism.
[0042] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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, including, but not limited to that represented by SEQ ID
NO:81, wherein the organism 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
organism.
[0043] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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, including, but not limited to that represented by SEQ ID
NO:81, wherein the organism 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 organism;
and wherein the organism 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.
[0044] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 including, but not limited to that represented by SEQ ID
NO:81, wherein the organism 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 organism;
wherein the organism contains an additional genetic modification to
express one or more heterologous proteins from an organism that
endogenously produces PUFAs; and wherein the protein utilizes
PUFA-CoA as substrates in forming phospholipids (PL) or
triacylglycerols (TAG).
[0045] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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, including, but not limited to that represented by SEQ ID
NO:81, wherein the organism 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 organism;
and wherein the organism comprises an additional genetic
modification to delete or inactivate an endogenous fatty acid
synthase (FAS) or protein associated with an FAS expressed by the
organism.
[0046] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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, including, but not limited to that represented by SEQ ID
NO:81, wherein the organism 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 organism;
wherein the organism 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; wherein the organism contains
an additional genetic modification to express one or more
heterologous proteins from an organism that endogenously produces
PUFAs; and wherein the protein utilizes PUFA-CoA as substrates in
forming phospholipids (PL) or triacylglycerols (TAG).
[0047] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 including, but not limited to that represented by SEQ ID
NO:81, wherein the organism 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 organism;
wherein the organism 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; and wherein the organism
comprises an additional genetic modification to delete or
inactivate an endogenous fatty acid synthase (FAS) or protein
associated with an FAS expressed by the organism.
[0048] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 including, but not limited to that represented by SEQ ID
NO:81, wherein the organism 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 organism;
wherein the organism contains an additional genetic modification to
express one or more heterologous proteins from an organism that
endogenously produces PUFAs; and wherein the protein utilizes
PUFA-CoA as substrates in forming phospholipids (PL) or
triacylglycerols (TAG); and wherein the organism comprises an
additional genetic modification to delete or inactivate an
endogenous fatty acid synthase (FAS) or protein associated with an
FAS expressed by the organism.
[0049] In another embodiment, the invention provides a genetically
modified organism, including a microorganism, plant, part of the
plant, or plant cell, wherein the organism 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 including, but not limited to that represented by SEQ ID
NO:81, wherein the organism 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 organism;
wherein the organism 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; wherein the organism contains
an additional genetic modification to express one or more
heterologous proteins from an organism that endogenously produces
PUFAs; and wherein the protein utilizes PUFA-CoA as substrates in
forming phospholipids (PL) or triacylglycerols (TAG); and wherein
the organism comprises an additional genetic modification to delete
or inactivate an endogenous fatty acid synthase (FAS) or protein
associated with an FAS expressed by the organism. In some
embodiments, the organism contains a genetic modification to
inhibit the expression or activity of one of the proteins KASII or
KASIII.
[0050] In other embodiments, the organism produces an increased
level of said at least one PUFA as compared to in the absence of
said inhibition of KASII or KASIII.
[0051] The genetic modification can comprise the transformation of
the organism with an RNAi construct that inhibits the expression or
activity of KASII, or an RNAi construct that inhibits the
expression or activity of KASIII. The RNAi construct can comprise a
nucleic acid sequence represented herein by SEQ ID NO:122 or by SEQ
ID NO:124.
[0052] In other embodiments, the genetic modification comprises the
transformation of the organism with an antisense nucleic acid
molecule that inhibits the expression or activity of KASII, or an
antisense nucleic acid molecule that inhibits the expression or
activity of KASIII. The antisense nucleic acid molecule can
comprises a nucleic acid sequence represented herein by SEQ ID
NO:123 or by SEQ ID NO:125.
[0053] In embodiments in which the organism 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,
the organism can be 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. In other
embodiments, the organism 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. In still other embodiments, the organism 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. In still other embodiments, the organism
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; and more
preferably, 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. In yet
further embodiments, the organism 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. In still further
embodiments, the organism 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.
[0054] In some embodiments wherein the organism contains an
additional genetic modification to express one or more heterologous
proteins from an organism that endogenously produces PUFAs; and
wherein the protein utilizes PUFA-CoA as substrates in forming
phospholipids (PL) or triacylglycerols (TAG), the organism
endogenously expresses a PUFA synthase. In other embodiments, the
protein is a DAGAT or an LPAAT. In other embodiments, the organism
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). In still other
embodiments, the organism 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). In some embodiments,
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. In
other embodiments, the organism 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; and more preferably 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. In still other
embodiments, the organism 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. In other embodiments, the organism 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. In wherein
the organism 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). In certain
embodiments, the organism 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.
[0055] In some embodiments of any of the foregoing embodiments, the
PUFA synthase comprises at least one functional domain from a PUFA
synthase from a Thraustochytrid or a Labyrinthulid. In some
embodiments, the PUFA synthase comprises at least one functional
domain from a PUFA synthase from a Thraustochytriales
microorganism. In other embodiments, 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. In still other
embodiments, 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 these microorganisms. In some embodiments,
the PUFA synthase comprises at least one functional domain from a
PUFA synthase from a marine bacterium. In other embodiments, 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. In still other
embodiments, the PUFA synthase consists of one or more proteins
comprising:
[0056] at least one enoyl-ACP reductase (ER) domain;
[0057] at least four acyl carrier protein (ACP) domains;
[0058] at least two .beta.-ketoacyl-ACP synthase (KS) domains;
[0059] at least one acyltransferase (AT) domain;
[0060] at least one .beta.-ketoacyl-ACP reductase (KR) domain;
[0061] at least two FabA-like .beta.-hydroxyacyl-ACP dehydrase (DH)
domains; and
[0062] at least one chain length factor (CLF) domain;
[0063] at least one malonyl-CoA:ACP acyltransferase (MAT)
domain.
[0064] In further embodiments, the PUFA synthase consists of one or
more proteins comprising:
[0065] two enoyl ACP-reductase (ER) domains;
[0066] eight or nine acyl carrier protein (ACP) domains;
[0067] two .beta.-keto acyl-ACP synthase (KS) domains;
[0068] one acyltransferase (AT) domain;
[0069] one ketoreductase (KR) domain;
[0070] two FabA-like .beta.-hydroxy acyl-ACP dehydrase (DH)
domains;
[0071] one chain length factor (CLF) domain; and
[0072] one malonyl-CoA:ACP acyltransferase (MAT) domain.
[0073] In still further embodiments, the PUFA synthase is a
bacterial PUFA synthase that produces PUFAs at temperatures of at
least about 25.degree. C., and wherein the PUFA synthase consists
of one or more proteins comprising:
[0074] at least one enoyl ACP-reductase (ER) domain;
[0075] at least six acyl carrier protein (ACP) domains;
[0076] at least two .beta.-keto acyl-ACP synthase (KS) domains;
[0077] at least one acyltransferase (AT) domain;
[0078] at least one ketoreductase (KR) domain;
[0079] at least two FabA-like .beta.-hydroxy acyl-ACP dehydrase
(DH) domains;
[0080] at least one chain length factor (CLF) domain;
[0081] at least one malonyl-CoA:ACP acyltransferase (MAT) domain;
and
[0082] at least one 4'-phosphopantetheinyl transferase (PPTase)
domain.
[0083] In some embodiments, 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.
[0084] In some embodiments, 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. In
other embodiments, expression of the PUFA synthase and the PPTase
is targeted to the plastid of the plant or plant cell.
[0085] In some embodiments, the genetically modified organism is a
plant and the plant is an oil seed plant. In other embodiments, the
plant is a dicotyledonous plant. In still other embodiments, the
plant is selected from, but is not limited to, the group consisting
of: canola, soybean, rapeseed, linseed, corn, safflower, sunflower
and tobacco.
[0086] In still other embodiments, the genetically modified
organism 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)), and any combinations
thereof. In some embodiments, the genetically modified organism
produces at least one polyunsaturated fatty acid (PUFA) selected
from the group consisting of: DHA, EPA and DPAn-6. In other
embodiments, the genetically modified organism produces DHA and
DPAn-6. In still other embodiments, the genetically modified
organism produces ARA.
[0087] In some embodiments, the genetically modified organism
comprises at least 0.5% by weight of said at least one PUFA. In
other embodiments, 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
organism. In still other embodiments, 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 organism.
[0088] In still further embodiments, 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.
[0089] In still further embodiments, 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.
[0090] In yet further embodiments, 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).
[0091] In other embodiments, the total fatty acid profile in the
genetically modified organism 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).
[0092] In other embodiments, the total fatty acid profile in the
genetically modified organism 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).
[0093] In other embodiments, the total fatty acid profile in the
genetically modified organism contains less than 0.5% by weight of
gamma-linolenic acid (GLA; 18:3, n-6).
[0094] The present invention also provides an oil obtained from any
of the genetically modified organisms of the invention. In one
embodiment, the invention provides an 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 any of the genetically modified organisms of the
invention.
[0095] Where the genetically modified organism is a plant, the
invention provides seeds obtained from the plant.
[0096] The invention also provides a food product comprising any
oil or seed of the present invention.
[0097] The invention also provides a pharmaceutical product that
contains an oil of the present invention.
[0098] The present invention also provides a method to produce an
oil comprising at least one PUFA, comprising recovering an oil from
a seed of the present invention.
[0099] The present invention also provides a method to produce an
oil comprising at least one PUFA, comprising recovering an oil from
any genetically modified organism of the present invention.
[0100] The present invention also provides a method to produce at
least one polyunsaturated fatty acid (PUFA), comprising growing any
genetically modified plant or microorganism of the present
invention.
[0101] The present invention further provides a method to provide a
supplement or therapeutic product containing at least one PUFA to
an individual, comprising providing to the individual a genetically
modified organism of the present invention, seeds of the present
invention, an oil of the present invention, a food product of the
present invention, or a pharmaceutical product of the present
invention.
[0102] The present invention also provides a method to produce the
foregoing genetically modified organisms, comprising transforming a
organism with one or more nucleic acid molecules encoding the PUFA
synthase and the PPTase, wherein the organism contains a genetic
modification to inhibit the expression or activity of a protein
selected from the group consisting of KASII and KASIII.
[0103] The present invention also provides a method to produce the
foregoing genetically modified organisms, comprising transforming a
organism with one or more nucleic acid molecules encoding the PUFA
synthase and the PPTase, and further genetically modifying the
organism to inhibit the expression or activity of a protein
selected from the group consisting of KASII and KASIII.
[0104] The invention also provides a process for transforming an
organism to express PUFAs, comprising transforming an organism with
nucleic acid molecules encoding a PUFA synthase, with a nucleic
acid molecule encoding a phosphopantetheinyl transferase (PPTase),
and with any of the acyl-CoA synthetase or acyltransferase
described herein. In one aspect, the organism contains a genetic
modification to delete or inactivate a fatty acid synthase (FAS)
expressed by the organism. In one aspect, the organism contains a
genetic modification to reduce competition for malonyl CoA with the
PUFA synthase or to increase the level of malonyl CoA in the
organism. The organism can include a plant or a microorganism, for
example.
BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION
[0105] FIG. 1 is a digitized image showing a phosphorimage analysis
of in vitro activity assays of cell free homogenates of
Schizochytrium strain Ac66 and PUFA-S KO and FAS KO mutants derived
from that strain.
[0106] FIG. 2 is a digitized image showing the phosphorimage
analysis of normal phase TLC separations of in vitro activity
assays in the Schizochytrium FAS-KO strain. Reactions were run for
the indicated times.
[0107] FIG. 3 is a digitized image showing the phosphorimage
analysis of normal phase TLC separations of in vitro activity
assays the Schizochytrium FAS-KO strain. Standard assay components
were used but the NADH, NADPH and acetyl-CoA components were varied
(Lane 1--NADH/NADPH/acetyl-CoA, Lane 2--NADPH/acetyl-CoA, Lane
3--NADH/acetyl-CoA, Lane 4--NADH/NADPH, Lane 5--none).
[0108] FIG. 4 is a digitized image showing the phosphorimage
analysis of normal phase TLC separations of in vitro activity
assays the Schizochytrium FAS-KO strain. Reactions were run for 10
minutes then ATP and Mg+2 were added. The reactions were stopped at
the times indicated at the bottom (''=sec, '=min).
[0109] FIG. 5 is a digitized image showing the phosphorimage
analysis of normal phase TLC separations of in vitro activity
assays the Schizochytrium FAS-KO strain. Reactions were run for 10
minutes, ATP and Mg+2 were added (except in sample 1) and
incubations continued for an additional 20 min (Lane 3--2 uL DMSO,
Lane 4--4 uL DMSO, Lane 5--25 uM Triascin C, Lane 6--100 uM
Triascin C, Lane 7--200 uM Triascin C).
[0110] FIG. 6A is a digital image showing the FAME analysis of E.
coli expressing Schizochytrium OrfA, OrfB*, OrfC and Het I. Target
PUFAs in the homogenate, high speed pellet fraction (P2),
supernatant fraction (S1) and high speed supernatant fraction (S2)
are shown.
[0111] FIG. 6B is a digital image showing the results of assays of
samples of the same E. coli strain used for FIG. 6A, except that
the lipid products were simply extracted with HIP (rather than
being converted to FAMES) prior to separation by TLC.
[0112] FIG. 7 is a FAME profile of control yeast and yeast
expressing Schizochytrium OrfsA, OrfsB, OrfC and Het I.
[0113] FIG. 8 is the FAME profile for yeast from FIG. 1, expanded
to illustrate the production of target PUFAs.
[0114] FIG. 9 is a graph showing the effects of inhibition of FAS
activity on DHA profiles (as a percentage of total FAME) of yeast
expressing Schizochytrium PUFA synthase (sOrfA, sOrfB, OrfC) and
Het I, alone or in combination with expression of an acyl CoA
synthetase.
[0115] FIG. 10 is a graph showing the effects of inhibition of FAS
activity on DHA and DPAn6 profiles (as a percentage of total FAME)
of yeast expressing Schizochytrium PUFA synthase (sOrfA, sOrfB,
OrfC) and Het I, alone or in combination with expression of an acyl
CoA synthetase.
[0116] FIG. 11 is a FAME profile showing the combined effects of
inhibition of FAS activity (by cerulenin), expression of
Schizochytrium PUFA synthase (sOrfA, sOrfB, OrfC) and Het I, and
expression of an acyl CoA synthetase, on DHA and DPAn6 production
in yeast.
[0117] FIG. 12 shows the lipid profile from a Schizochytrium in
which a DAGAT gene has been knocked out.
[0118] FIG. 13 is a FAME profile of wild-type Arabidopsis and
Arabidopsis Line 263 (plastid targeted), expressing Schizochytrium
Orfs A, B*, C and Het I during seed development.
[0119] FIG. 14 is a FAME profile of an Arabidopsis seed from Line
1087-7 (plastid targeted), expressing Schizochytrium Orfs A, B*, C
and HetI targeted to the plastid combined with FAS inhibition (KAS
III antisense) during seed development.
[0120] FIG. 15 is a FAME profile of pooled Arabidopsis seed from
Line 1366 expressing Schizochytrium Orfs A, B*, C and HetI targeted
to the plastid combined with FAS inhibition (KAS II RNAi) and ACS-1
during seed development.
DETAILED DESCRIPTION OF THE INVENTION
[0121] The present invention generally relates to the provision of
proteins or targets (generally referred to herein as "accessory
proteins" or "accessory targets"), and nucleic acid molecules
encoding such proteins, for the improvement of the production of
polyunsaturated fatty acids (PUFAs) and particularly, long chain
PUFAs (LCPUFAs), in a host organism that has been genetically
modified to produce such PUFAs. The present invention also relates
to the organisms that have been genetically modified to express
certain of such proteins, and to methods of making and using such
proteins and organisms. The present invention also relates to
additional genetic modifications to organisms that produce PUFAs
(including by genetic modification to produce PUFAs), which can
include deletions or inactivations of particular genes or targets
in the organism. In particular, the present invention relates to
the genetic modification of organisms that express a PUFA PKS
system (either endogenously or by genetic manipulation), to improve
or enhance PUFA production and/or accumulation by the organism. For
example, the present invention also relates to engineering the down
regulation of enzymes that compete for substrate and to the
engineering of higher enzyme activities such as by mutagenesis, or
targeting of enzymes to plastid organelles, as well as the
cytosol.
[0122] According to the present invention, an organism that has
been genetically modified to express a PUFA PKS system (also known
as a PUFA synthase system, which is used interchangeably with PUFA
PKS system or PKS-like system for the production of PUFAs), wherein
the organism does not naturally (endogenously, without genetic
modification) express such a system, or at least that particular
PUFA PKS system or portion thereof with which the organism is being
genetically modified, can be referred to herein as a "heterologous"
host organism with regard to the modification of the organism with
the PUFA PKS system or with another protein that is not
endogenously expressed by the organism. The genetic modifications
of the present invention may also be used to improve PUFA
production in a host organism that endogenously expresses a PUFA
PKS system, where the organism is not further modified with a
different PUFA PKS system or a portion thereof.
[0123] More particularly, the present inventors have discovered and
disclose for the first time herein that the fatty acid products of
the Schizochytrium PUFA synthase (primarily DHA and DPAn-6) are
released from that enzyme as free fatty acids (FFA), and that the
release mechanism is integral to the enzyme. This product release
mechanism is believed to be a characteristic of all thraustochytrid
PUFA PKS (PUFA synthase) enzyme systems, and may be a
characteristic of all eukaryotic PUFA PKS systems, including
labyrinthulid systems. Further, the present inventors show, using
Schizochytrium as a model, that the DHA and DPA FFAs are
subsequently esterified to coenzyme A (CoA) by the action of an
endogenous acyl-CoA synthetase (ACoAS or ACS) or synthetases. These
activated forms of fatty acids (acyl-CoAs) can then serve as the
substrates for PL and TAG forming enzymes.
[0124] The endogenous enzymes of Schizochytrium are very efficient
in converting the FFA products of its PUFA synthase into acyl-CoA
and then using those for PL and TAG synthesis. This is evidenced by
the high level of DHA and DPA accumulation in Schizochytrium oil
and PL fractions. However, without being bound by theory, the
present inventors believe that the ACoAS enzymes present in
heterologous hosts into which PUFA synthase systems can be
transformed may not carry out those reactions as efficiently as do
the ACoAS from the PUFA synthase donor organism. Additionally, the
endogenous acyl-transferase enzymes which form PL and TAG in those
new host organisms may not efficiently utilize PUFA-CoA as
substrates, particularly as compared to the organism from which the
PUFA synthase was derived. The inventors also propose that
acyltransferases from certain organisms may be generally better
enzymes for accumulation of PUFAs in the oil and oil fractions of
host organisms, especially certain PUFAs, than similar enzymes from
other organisms (e.g., an acyltransferase from one organism may
transfer more DHA-CoA units into a TAG than an acyltransferase from
a different organism). Therefore, the present inventors disclose
herein that an organism like Schizochytrium, but not limited to
Schizochytrium, (e.g., a thraustochytrid or another organism, and
particularly another eukaryotic organism), which produces its PUFAs
via a PUFA synthase enzyme (PUFA PKS system) or through another
acyl chain biosynthesis system, and which accumulates high levels
of PUFA in its PL and TAG, will serve as a good source of genes
encoding those enzymes.
[0125] The discovery by the present inventors of the release of the
PUFA product from the PUFA synthase as a FFA represents both
challenges and opportunities in terms of transferring the system to
heterologous hosts, and provides substantial opportunity to control
and improve the efficiency of production of PUFAs in a heterologous
host organism.
[0126] By way of explanation, long chain PUFAs (LCPUFAs) do not
occur as FFAs as a part of the "standard" or "classical" PUFA
biosynthetic pathway (defined below). In fact, organisms will
usually only encounter a PUFA as a FFA is when it is provided
exogenously. For example, E. coli, like most bacteria, does not
synthesize PUFAs. The 16 and 18 carbon saturated or
mono-unsaturated fatty acids produced by these organisms are
synthesized on acyl carrier proteins (ACPs) via a Type II FAS
system. The acyl-ACPs serve as substrates for the PL forming
enzymes. E. coli can utilize a variety of FFAs as exogenous carbon
sources. Those FFAs are converted to acyl-CoA prior to their entry
into PLs or into a degradation cycle. The FadD gene encodes the
only known ACoAS enzyme in E. coli, and mutations in that gene
result in the inability to grow on FFAs as the sole carbon
source.
[0127] Eukaryotic organisms typically produce saturated fatty acids
(16 and 18 carbon) using a Type I fatty acid synthase (FAS) (or a
Type II FAS in the case of higher plants). The products of the FAS
system can be released as FFA (e.g. animal FAS) or as acyl-CoAs
(e.g. fungal FAS). In the case of plants, the Type II FAS is
localized in plastids. In this case, 16 or 18 carbon fatty acids
are produced via the Type II FAS and often, a single double bond is
formed while that fatty acid is attached to ACP. The acyl-ACPs can
serve as substrates for formation of plastidial PL. For those fatty
acids destined for export from the plastid (for use in cytoplasmic
PL or for TAG synthesis), an acyl-ACP thioesterase hydrolyzes the
thioester bond to release a FFA. The FFA is then exported from the
plastid and converted to an acyl-CoA by a cytoplasmic ACoAS. These
acyl-CoAs serve as the substrates for PL and TAG synthesis
enzymes.
[0128] The "standard" or "classical" pathway for synthesis of long
chain PUFAs (LCPUFAs) in eukaryotic organisms involves the
modification of medium chain-length saturated or mono-unsaturated
fatty acids (e.g., the products of the FAS system described above).
These modifications consist of elongation steps and desaturation
steps. The substrates for the elongation reaction are fatty
acyl-CoA (the fatty acid chain to be elongated) and malonyl-CoA
(the source of the two carbons added during each elongation
reaction). The product of the elongase reaction is a fatty acyl-CoA
that has two additional carbons in the linear chain. Free fatty
acids (FFAs) do not normally occur in this reaction cycle. The
desaturases create cis double bonds in the preexisting fatty acid
chain by extraction of two hydrogens in an oxygen-dependant
reaction. The substrates for the desaturases are either acyl-CoAs
(in some animals) or fatty acids that are esterified to the
glycerol backbone of a PL (e.g., phosphotidylcholine). Again, FFAs
do not occur in this reaction mechanism. Therefore, the only time
FFAs occur in "standard" or "classical" LCPUFA synthesis pathways
is during release of the fatty acids from some FAS systems. As
discussed above, these are typically 16 or 18 carbon fatty acids
and usually are either saturated or monounsaturated fatty acids,
not longer chain PUFAs such as EPA or DHA. One consequence of this
scheme for long chain PUFA production is that intermediates in the
pathway often accumulate, often representing the majority of the
novel fatty acids produced by the system.
[0129] Therefore, according to the present invention, reference to
a "standard" or "classical" pathway for the production of PUFAs
refers to the fatty acid synthesis pathway where medium
chain-length saturated fatty acids (e.g., products of a fatty acid
synthase (FAS) system) are modified by a series of elongation and
desaturation reactions. The substrates for the elongation reaction
are fatty acyl-CoA (the fatty acid chain to be elongated) and
malonyl-CoA (the source of the 2 carbons added during each
elongation reaction). The product of the elongase reaction is a
fatty acyl-CoA that has two additional carbons in the linear chain.
The desaturases create cis double bonds in the preexisting fatty
acid chain by extraction of 2 hydrogens in an oxygen-dependant
reaction. Such pathways and the genes involved in such pathways are
well-known in the literature (e.g., see Background).
[0130] The pathway for synthesis of long chain PUFAs via the PUFA
PKS (PUFA synthase) enzymes (described in detail below) is very
different from the "standard" pathway described above. The PUFA
synthases utilize malonyl-CoA as a carbon source and produce the
final PUFA without releasing intermediates in any significant
amount. The appropriate cis double bonds are added during the
synthesis using a mechanism that does not require oxygen. NADPH is
used as a reductant during the synthesis cycles. In at least
Thraustochytrid PUFA PKS systems, the enzymes release the PUFA
product as a FFA, as has been disclosed for the first time by the
present inventors herein. This release mechanism is part of the
enzyme itself. Therefore, the release of LCPUFAs as FFA from the
PUFA enzyme system is a unique feature of the PUFA PKS system of
Schizochytrium and is likely to be a feature of all eukaryotic PUFA
synthase systems such as those in thraustochytrids.
[0131] Accordingly, the present inventors propose that, when
expressing a PUFA PKS system (PUFA synthase system) in a
heterologous host (e.g., a host organism that does not endogenously
express that particular PUFA PKS system), a factor to consider with
regard to optimizing the PUFA production and accumulation in the
desired compartments or lipid fractions is the ability of that
host's endogenous acyl-CoA synthetase (ACoAS) enzyme(s) to
recognize the FFA product of the introduced system as a substrate
for conversion to the corresponding acyl-CoA. Since, as discussed
above, most heterologous host organisms into which a PUFA PKS
system may be introduced usually only encounter a PUFA as an FFA
when it is provided exogenously, the host organism may not have
optimal accessory proteins in place to handle the FFAs, which can
present an inhibitory factor in the optimal production and
accumulation of PUFAs in a desired lipid fraction or compartment by
a host organism. For example, it is well known that there are
several families of proteins that have ACoAS activity, and that the
FFA substrate preferences of these enzymes can be fairly specific.
Therefore, the ACoASs present in some potential hosts may not
efficiently convert long chain PUFA FFA to acyl-CoA, particularly
if those hosts do not normally encounter the FFA forms of those
PUFA. In addition, a host organism may not have optimal
acyltransferases that form PL and TAG and are able to utilize the
PUFA-CoA as substrates. Finally, even in host organisms that
endogenously express a PUFA PKS system, the present inventors
believe that it is possible to genetically modify the organism
using the modifications discussed herein to improve the
accumulation of PUFAs in the oils and oil fractions in the
organism.
[0132] The pathway and discoveries by the present inventors
described above provides several guidelines or strategies for the
production of PUFAs in heterologous (or native) hosts by expression
of a PUFA synthase:
[0133] 1. Gene Optimization Optimization of the genes sequences to
match those of the heterologous host may be needed in order to
obtain expression of the proteins. This is illustrated in the
Examples described below, where genes encoding proteins from a PUFA
PKS system from Schizochytrium are optimized for codon usage in a
bacterial host as well as yeast. A gene optimized for use in
bacteria was also found to be useful for expression of the
Schizochytrium PUFA PKS in plants. Details regarding these
optimized genes are described below.
[0134] 2. PPTase Expression The present inventors have determined
that endogenous PPTases present in E. coli, yeast and plants are
not able to activate the PUFA synthase ACP domains. The present
inventors have previously identified a suitable alternative PPTase,
Het I from Nostoc (described in U.S. Patent Application Publication
No. 20020194641), which can be used in hosts whose endogenous
PPTases do not activate the PUFA synthase ACP domains. Other
suitable PPTases are also described and can be readily obtained.
Use of PPTases in a variety of heterologous host cells is described
and exemplified below.
[0135] 3. Modification of Substrate Flux/Inhibition of FAS PUFA
synthases utilize malonyl-CoA as the source of carbon for
elongation reactions. Malonyl-CoA is also used by FASs, cytoplasmic
fatty acid elongation reactions and other enzymes (e.g., chalcone
synthase). The PUFA synthase competes with these other enzyme
systems for the malonyl-CoA. This indicates that one way to
increase the flux through the PUFA synthase pathway would be to
enhance its ability to compete for the malonyl-CoA pool(s). There
are many possible ways to achieve enhanced ability to compete for
this substrate. These include, but are not limited to, 1)
inhibition of competing pathways, including inhibition of any
elements in the FAS pathway, e.g., by reducing expression levels of
enzymes or subunits involved in those pathways (e.g., by use of
antisense RNA, RNAi, co-suppression, or mutations), 2) expression
of the PUFA synthase in heterologous hosts in which competing
pathways have been reduced or blocked (e.g., in Canola where the
ability to elongate fatty acids in the cytoplasm has been blocked),
and/or 3) by increasing the pool of malonyl-CoA (e.g., by
expression of acetyl-CoA carboxylase). Examples of this strategy
are described in more detail below and illustrated in the
Examples.
[0136] 4. Expression of Acyl-CoA Synthetases Enzymes present in
Schizochytrium efficiently convert the free fatty acid products of
the PUFA synthase to acyl-CoA. Enzymes present in heterologous
hosts may not carry out these reactions with similar efficiency
since those free fatty acids may not typically be encountered by
those organisms. For example, expression of acyl-CoA synthetase
enzymes that efficiently convert the free fatty acid products of
the various PUFA synthases (e.g., DHA, DPA n-6, EPA, or other
products) to acyl-CoA in those heterologous hosts may result in the
increased ability to accumulate those products. In this regard,
Schizochytrium, or other organisms that produce PUFAs via the PUFA
synthase pathway, will serve as a good source of genes encoding
those enzymes (see description and Examples below).
[0137] 5. Expression of Acyltransferases and Related Enzymes
Enzymes present in Schizochytrium efficiently utilize the acyl-CoA
forms of the products of the PUFA synthase to synthesize PL and TAG
molecules. Enzymes present in heterologous hosts may not carry out
these reactions with similar efficiency since those PUFA-CoAs may
not typically be encountered by those organisms. For example,
expression of PL or TAG synthesis enzymes that efficiently
integrate the acyl-CoA products of the various PUFA synthases
(e.g., DHA-CoA, DPA n-6-CoA, EPA-CoA, or others) into PL or TAG
molecules in those heterologous hosts may result in the increased
ability to accumulate those products. In this regard,
Schizochytrium, or other organisms that produce PUFAs via the PUFA
synthase pathway, will serve as a good source of genes encoding
those enzymes (see description and Examples below).
[0138] 6. Organelle-specific Expression Other methods are
envisioned herein that can be utilized to increase the amount, or
alter the profile, of PUFA accumulating in heterologous hosts. As
one example, one can express the PUFA synthase system in separate
compartments in the host, thereby accessing separate malonyl-CoA
pools, which may result in increased accumulation (e.g., in the
plastid and cytoplasm of plant cells). This strategy is also
exemplified in the Examples below.
[0139] Accordingly, the present invention provides a solution to
the potential inhibition of PUFA production and/or accumulation in
heterologous host organisms and also provides a unique opportunity
to control and enhance the production of PUFAs in any organism that
produces PUFAs using a PUFA PKS system (either by genetic
modification or endogenously). Specifically, the present invention
provides various targets in the form of proteins and nucleic acid
molecules encoding such proteins that can be expressed in organisms
that have been genetically modified to express a PUFA PKS system,
as well as other genetic modifications and strategies described
herein, in order to enhance or increase the production and/or
accumulation of PUFAs by the organism, particularly in desired
compartments or lipid fractions in the organism. Such targets can
generally be referred to herein as "accessory" targets for a PUFA
PKS system. As used herein, a target can represent a nucleic acid
molecule and/or its encoded protein for which expression or
overexpression is desired in a host organism as described herein,
as well as a target for deletion or inactivation, or even a target
organelle (e.g., targeting to the plastid of a plant). In other
words, a target can be element added to or any modification of an
enzyme system for the production of PUFAs, and particularly a PUFA
PKS system, wherein the target is identified as useful with respect
to the increased or improved production and/or accumulation of
fatty acids in a host organism.
PUFA PKS Systems (PUFA Synthases)
[0140] Accordingly, the present invention is directed to the
provision of accessory proteins and other targets for use in
connection with a PUFA PKS system. As used herein, a PUFA PKS
system (which may also be referred to as a PUFA synthase system or
PUFA synthase) generally has the following identifying features:
(1) it produces PUFAs, and particularly, long chain PUFAs, as a
natural product of the system; and (2) it comprises several
multifunctional proteins assembled into a complex that conducts
both iterative processing of the fatty acid chain as well
non-iterative processing, including trans-cis isomerization and
enoyl reduction reactions in selected cycles. In addition, the ACP
domains present in the PUFA synthase enzymes require activation by
attachment of a cofactor (4-phosphopantetheine). Attachment of this
cofactor is carried out by phosphopantetheinyl transferases
(PPTase). If the endogenous PPTases of the host organism are
incapable of activating the PUFA synthase ACP domains, then it is
necessary to provide a PPTase that is capable of carrying out that
function. The inventors have identified the Het I enzyme of Nostoc
sp. as an exemplary and suitable PPTase for activating PUFA
synthase ACP domains. Reference to a PUFA PKS system or a PUFA
synthase refers collectively to all of the genes and their encoded
products that work in a complex to produce PUFAs in an organism.
Therefore, the PUFA PKS system refers specifically to a PKS system
for which the natural products are PUFAs.
[0141] More specifically, a PUFA PKS system as referenced herein
produces polyunsaturated fatty acids (PUFAs) and particularly, long
chain PUFAs (LCPUFAs), as products. For example, an organism that
endogenously (naturally) contains a PUFA PKS system makes PUFAs
using this system. According to the present invention, PUFAs are
fatty acids with a carbon chain length of at least 16 carbons, and
more preferably at least 18 carbons, and more preferably at least
20 carbons, and more preferably 22 or more carbons, with at least 3
or more double bonds, and preferably 4 or more, and more preferably
5 or more, and even more preferably 6 or more double bonds, wherein
all double bonds are in the cis configuration. Reference to long
chain polyunsaturated fatty acids (LCPUFAs) herein more
particularly refers to fatty acids of 18 and more carbon chain
length, and preferably 20 and more carbon chain length, containing
3 or more double bonds. LCPUFAs of the omega-6 series include:
gamma-linolenic acid (C18:3), di-homo-gamma-linolenic acid
(C20:3n-6), arachidonic acid (C20:4n-6), adrenic acid (also called
docosatetraenoic acid or DTA) (C22:4n-6), and docosapentaenoic acid
(C22:5n-6). The LCPUFAs of the omega-3 series include:
alpha-linolenic acid (C18:3), eicosatrienoic acid (C20:3n-3),
eicosatetraenoic acid (C20:4n-3), eicosapentaenoic acid (C20:5n-3),
docosapentaenoic acid (C22:5n-3), and docosahexaenoic acid
(C22:6n-3). The LCPUFAs also include fatty acids with greater than
22 carbons and 4 or more double bonds including but not limited to
C28:8(n-3).
[0142] A PUFA PKS system according to the present invention also
comprises several multifunctional proteins (and can include single
function proteins, particularly for PUFA PKS systems from marine
bacteria) that are assembled into a complex that conducts both
iterative processing of the fatty acid chain as well non-iterative
processing, including trans-cis isomerization and enoyl reduction
reactions in selected cycles. These proteins can also be referred
to herein as the core PUFA PKS enzyme complex or the core PUFA PKS
system. The general functions of the domains and motifs contained
within these proteins are individually known in the art and have
been described in detail with regard to various PUFA PKS systems
from marine bacteria and eukaryotic organisms (see, e.g., U.S. Pat.
No. 6,140,486; U.S. Pat. No. 6,566,583; Metz et al., Science
293:290-293 (2001); U.S. Patent Application Publication No.
20020194641; U.S. Patent Application Publication No. 20040235127;
U.S. Patent Application Publication No. 20050100995, and PCT
Publication No. WO 2006/135866). The domains may be found as a
single protein (i.e., the domain and protein are synonymous) or as
one of two or more (multiple) domains in a single protein, as
mentioned above.
[0143] The domain architecture of various PUFA PKS systems from
marine bacteria and members of Thraustochytrium, and the structural
and functional characteristics of genes and proteins comprising
such PUFA PKS systems, have been described in detail (see, e.g.,
U.S. Pat. No. 6,140,486; U.S. Pat. No. 6,566,583; Metz et al.,
Science 293:290-293 (2001); U.S. Patent Application Publication No.
20020194641; U.S. Patent Application Publication No. 20040235127;
U.S. Patent Application Publication No. 20050100995 and PCT
Publication No. WO 2006/135866).
[0144] PUFA PKS systems and proteins or domains thereof that are
useful in the present invention include both bacterial and
non-bacterial PUFA PKS systems. A non-bacterial PUFA PKS system is
a PUFA PKS system that is from or derived from an organism that is
not a bacterium, such as a eukaryote or an archaebacterium.
Eukaryotes are separated from prokaryotes based on the degree of
differentiation of the cells, with eukaryotes being more
differentiated than prokaryotes. In general, prokaryotes do not
possess a nuclear membrane, do not exhibit mitosis during cell
division, have only one chromosome, contain 70S ribosomes in their
cytoplasm, do not possess mitochondria, endoplasmic reticulum,
chloroplasts, lysosomes or Golgi apparatus, and may have flagella,
which if present, contain a single fibril. In contrast, eukaryotes
have a nuclear membrane, exhibit mitosis during cell division, have
many chromosomes, contain 80S ribosomes in their cytoplasm, possess
mitochondria, endoplasmic reticulum, chloroplasts (in algae),
lysosomes and Golgi apparatus, and may have flagella, which if
present, contain many fibrils. In general, bacteria are
prokaryotes, while algae, fungi, protist, protozoa and higher
plants are eukaryotes. According to the present invention,
genetically modified plants can be produced which incorporate
non-bacterial PUFA PKS functional domains with bacterial PUFA PKS
functional domains, as well as PKS functional domains or proteins
from other PKS systems (Type I iterative or modular, Type II, or
Type III) or FAS systems.
[0145] Preferably, a PUFA PKS system of the present invention
comprises at least the following biologically active domains that
are typically contained on three or more proteins: (a) at least one
enoyl-ACP reductase (ER) domain; (b) multiple acyl carrier protein
(ACP) domain(s) (e.g., at least from one to four, and preferably at
least five ACP domains, and in some embodiments up to six, seven,
eight, nine, ten, or more than ten ACP domains); (c) at least two
.beta.-ketoacyl-ACP synthase (KS) domains; (d) at least one
acyltransferase (AT) domain; (e) at least one .beta.-ketoacyl-ACP
reductase (KR) domain; (f) at least two FabA-like
.beta.-hydroxyacyl-ACP dehydrase (DH) domains; (g) at least one
chain length factor (CLF) domain; (h) at least one malonyl-CoA:ACP
acyltransferase (MAT) domain. In one embodiment, a PUFA PKS system
according to the present invention also comprises at least one
region containing a dehydratase (DH) conserved active site
motif.
[0146] In a preferred embodiment, a PUFA PKS system comprises at
least the following biologically active domains: (a) at least one
enoyl-ACP reductase (ER) domain; (b) at least five acyl carrier
protein (ACP) domains; (c) at least two .beta.-ketoacyl-ACP
synthase (KS) domains; (d) at least one acyltransferase (AT)
domain; (e) at least one .beta.-ketoacyl-ACP reductase (KR) domain;
(f) at least two FabA-like .beta.-hydroxyacyl-ACP dehydrase (DH)
domains; (g) at least one chain length factor (CLF) domain; and (h)
at least one malonyl-CoA:ACP acyltransferase (MAT) domain. In one
embodiment, a PUFA PKS system according to the present invention
also comprises at least one region or domain containing a
dehydratase (DH) conserved active site motif that is not a part of
a FabA-like DH domain. The structural and functional
characteristics of each of these domains are described in detail in
U.S. Patent Application Publication No. 20020194641; U.S. Patent
Application Publication No. 20040235127; U.S. Patent Application
Publication No. 20050100995; and PCT Publication No. WO
2006/135866.
[0147] According to the present invention, a domain or protein
having 3-keto acyl-ACP synthase (KS) biological activity (function)
is characterized as the enzyme that carries out the initial step of
the FAS (and PKS) elongation reaction cycle. The term
".beta.-ketoacyl-ACP synthase" can be used interchangeably with the
terms "3-keto acyl-ACP synthase", ".beta.-keto acyl-ACP synthase",
and "keto-acyl ACP synthase", and similar derivatives. The acyl
group destined for elongation is linked to a cysteine residue at
the active site of the enzyme by a thioester bond. In the
multi-step reaction, the acyl-enzyme undergoes condensation with
malonyl-ACP to form -keto acyl-ACP, CO.sub.2 and free enzyme. The
KS plays a key role in the elongation cycle and in many systems has
been shown to possess greater substrate specificity than other
enzymes of the reaction cycle. For example, E. coli has three
distinct KS enzymes--each with its own particular role in the
physiology of the organism (Magnuson et al., Microbiol. Rev. 57,
522 (1993)). The two KS domains of the PUFA-PKS systems described
in marine bacteria and the thraustochytrids described herein may
have distinct roles in the PUFA biosynthetic reaction sequence. As
a class of enzymes, KS's have been well characterized. The
sequences of many verified KS genes are known, the active site
motifs have been identified and the crystal structures of several
have been determined. Proteins (or domains of proteins) can be
readily identified as belonging to the KS family of enzymes by
homology to known KS sequences.
[0148] According to the present invention, a domain or protein
having malonyl-CoA:ACP acyltransferase (MAT) biological activity
(function) is characterized as one that transfers the malonyl
moiety from malonyl-CoA to ACP. The term "malonyl-CoA:ACP
acyltransferase" can be used interchangeably with "malonyl
acyltransferase" and similar derivatives. In addition to the active
site motif (GxSxG), these enzymes possess an extended motif of R
and Q amino acids in key positions that identifies them as MAT
enzymes (e.g., in contrast to an AT domain described below). In
some PKS systems (but not the PUFA PKS domain) MAT domains will
preferentially load methyl- or ethyl-malonate on to the ACP group
(from the corresponding CoA ester), thereby introducing branches
into the linear carbon chain. MAT domains can be recognized by
their homology to known MAT sequences and by their extended motif
structure.
[0149] According to the present invention, a domain or protein
having acyl carrier protein (ACP) biological activity (function) is
characterized as being small polypeptides (typically, 80 to 100
amino acids long), that function as carriers for growing fatty acyl
chains via a thioester linkage to a covalently bound co-factor of
the protein. They occur as separate units or as domains within
larger proteins. ACPs are converted from inactive apo-forms to
functional holo-forms by transfer of the phosphopantetheinyl moiety
of CoA to a highly conserved serine residue of the ACP. Acyl groups
are attached to ACP by a thioester linkage at the free terminus of
the phosphopantetheinyl moiety. ACPs can be identified by labeling
with radioactive pantetheine and by sequence homology to known
ACPs. The presence of variations of the above mentioned motif
(LGIDS*) is also a signature of an ACP.
[0150] According to the present invention, a domain or protein
having ketoreductase activity, also referred to as 3-ketoacyl-ACP
reductase (KR) biological activity (function), is characterized as
one that catalyzes the pyridine-nucleotide-dependent reduction of
3-keto acyl forms of ACP. It is the first reductive step in the de
novo fatty acid biosynthesis elongation cycle and a reaction often
performed in polyketide biosynthesis. The term "3-ketoacyl-ACP
reductase" can be used interchangeably with the terms
"ketoreductase", "3-ketoacyl-ACP reductase", "keto-acyl ACP
reductase" and similar derivatives of the term. Significant
sequence similarity is observed with one family of enoyl ACP
reductases (ER), the other reductase of FAS (but not the ER family
present in the PUFA PKS systems), and the short-chain alcohol
dehydrogenase family. Pfam analysis of the PUFA PKS region
indicated above reveals the homology to the short-chain alcohol
dehydrogenase family in the core region. Blast analysis of the same
region reveals matches in the core area to known KR enzymes as well
as an extended region of homology to domains from the other
characterized PUFA PKS systems.
[0151] According to the present invention, a domain or protein is
referred to as a chain length factor (CLF) based on the following
rationale. The CLF was originally described as characteristic of
Type II (dissociated enzymes) PKS systems and was hypothesized to
play a role in determining the number of elongation cycles, and
hence the chain length, of the end product. CLF amino acid
sequences show homology to KS domains (and are thought to form
heterodimers with a KS protein), but they lack the active site
cysteine. CLF's role in PKS systems has been controversial. New
evidence (C. Bisang et al., Nature 401, 502 (1999)) suggests a role
in priming (providing the initial acyl group to be elongated) the
PKS systems. In this role the CLF domain is thought to
decarboxylate malonate (as malonyl-ACP), thus forming an acetate
group that can be transferred to the KS active site. This acetate
therefore acts as the `priming` molecule that can undergo the
initial elongation (condensation) reaction. Homologues of the Type
II CLF have been identified as `loading` domains in some modular
PKS systems. A domain with the sequence features of the CLF is
found in all currently identified PUFA PKS systems and in each case
is found as part of a multidomain protein.
[0152] An "acyltransferase" or "AT" refers to a general class of
enzymes that can carry out a number of distinct acyl transfer
reactions. The term "acyltransferase" can be used interchangeably
with the term "acyl transferase". The AT domains identified in the
PUFA PKS systems described herein show good homology one another
and to domains present in all of the other PUFA PKS systems
currently examined and very weak homology to some acyltransferases
whose specific functions have been identified (e.g. to
malonyl-CoA:ACP acyltransferase, MAT). In spite of the weak
homology to MAT, this AT domain is not believed to function as a
MAT because it does not possess an extended motif structure
characteristic of such enzymes (see MAT domain description, above).
For the purposes of this disclosure, the possible functions of the
AT domain in a PUFA PKS system include, but are not limited to:
transfer of the fatty acyl group from the ORFA ACP domain(s) to
water (i.e. a thioesterase--releasing the fatty acyl group as a
free fatty acid), transfer of a fatty acyl group to an acceptor
such as CoA, transfer of the acyl group among the various ACP
domains, or transfer of the fatty acyl group to a lipophilic
acceptor molecule (e.g. to lysophosphadic acid).
[0153] According to the present invention, this domain has enoyl
reductase (ER) biological activity. The ER enzyme reduces the
trans-double bond (introduced by the DH activity) in the fatty
acyl-ACP, resulting in fully saturating those carbons. The ER
domain in the PUFA-PKS shows homology to a newly characterized
family of ER enzymes (Heath et al., Nature 406, 145 (2000)). Heath
and Rock identified this new class of ER enzymes by cloning a gene
of interest from Streptococcus pneumoniae, purifying a protein
expressed from that gene, and showing that it had ER activity in an
in vitro assay. All of the PUFA PKS systems currently examined
contain at least one domain with very high sequence homology to the
Schizochytrium ER domain, which shows homology to the S. pneumoniae
ER protein.
[0154] According to the present invention, a protein or domain
having dehydrase or dehydratase (DH) activity catalyzes a
dehydration reaction. As used generally herein, reference to DH
activity typically refers to FabA-like .beta.-hydroxyacyl-ACP
dehydrase (DH) biological activity. FabA-like
.beta.-hydroxyacyl-ACP dehydrase (DH) biological activity removes
HOH from a .beta.-ketoacyl-ACP and initially produces a trans
double bond in the carbon chain. The term "FabA-like
.beta.-hydroxyacyl-ACP dehydrase" can be used interchangeably with
the terms "FabA-like .beta.-hydroxy acyl-ACP dehydrase",
".beta.-hydroxyacyl-ACP dehydrase", "dehydrase" and similar
derivatives. The DH domains of the PUFA PKS systems show homology
to bacterial DH enzymes associated with their FAS systems (rather
than to the DH domains of other PKS systems). A subset of bacterial
DH's, the FabA-like DH's, possesses cis-trans isomerase activity
(Heath et al., J. Biol. Chem., 271, 27795 (1996)). It is the
homology to the FabA-like DH proteins that indicate that one or all
of the DH domains described herein is responsible for insertion of
the cis double bonds in the PUFA PKS products.
[0155] A PUFA PKS protein useful of the invention may also have
dehydratase activity that is not characterized as FabA-like (e.g.,
the cis-trans activity described above is associated with FabA-like
activity), generally referred to herein as non-FabA-like DH
activity, or non-FabA-like .beta.-hydroxyacyl-ACP dehydrase (DH)
biological activity. More specifically, a conserved active site
motif (.about.13 amino acids long: L*xxHxxxGxxxxP; e.g.,
illustrated by amino acids 2504-2516 of SEQ ID NO:70; *in the
motif, L can also be I) is found in dehydratase domains in PKS
systems (Donadio S, Katz L. Gene. 1992 Feb. 1; 111(1):51-60). This
conserved motif, also referred to herein as a dehydratase (DH)
conserved active site motif or DH motif, is found in a similar
region of all known PUFA-PKS sequences described to date and in the
PUFA PKS sequences described herein, but it is believed that his
motif has only recently been detected. This conserved motif is
within an uncharacterized region of high homology in the PUFA-PKS
sequence. The proposed biosynthesis of PUFAs via the PUFA-PKS
requires a non-FabA like dehydration, and this motif may be
responsible for the reaction.
[0156] For purposes of illustration, the structure of several PUFA
PKS systems is described in detail below. However, it is to be
understood that this invention is not limited to the use of these
PUFA PKS systems.
Schizochytrium PUFA PKS System
[0157] In one embodiment, a PUFA PKS system from Schizochytrium
comprises at least the following biologically active domains: (a)
two enoyl-ACP reductase (ER) domain; (b) between five and ten or
more acyl carrier protein (ACP) domains, and in one aspect, nine
ACP domains; (c) two .beta.-ketoacyl-ACP synthase (KS) domains; (d)
one acyltransferase (AT) domain; (e) one .beta.-ketoacyl-ACP
reductase (KR) domain; (f) two FabA-like .beta.-hydroxyacyl-ACP
dehydrase (DH) domains; (g) one chain length factor (CLF) domain;
and (h) one malonyl-CoA:ACP acyltransferase (MAT) domain. In one
embodiment, a Schizochytrium PUFA PKS system according to the
present invention also comprises at least one region or domain
containing a dehydratase (DH) conserved active site motif that is
not a part of a FabA-like DH domain. The structural and functional
characteristics of these domains are generally individually known
in the art (see, e.g., U.S. Pat. No. 6,566,583; Metz et al.,
Science 293:290-293 (2001); U.S. Patent Application Publication No.
20020194641; and PCT Publication No. WO 2006/135866).
[0158] There are three open reading frames that form the core
Schizochytrium PUFA PKS system described previously. The domain
structure of each open reading frame is as follows.
Schizochytrium Open Reading Frame A (OrfA):
[0159] The complete nucleotide sequence for OrfA is represented
herein as SEQ ID NO:1. OrfA is a 8730 nucleotide sequence (not
including the stop codon) which encodes a 2910 amino acid sequence,
represented herein as SEQ ID NO:2. Within OrfA are twelve domains:
(a) one .beta.-keto acyl-ACP synthase (KS) domain; (b) one
malonyl-CoA:ACP acyltransferase (MAT) domain; (c) nine acyl carrier
protein (ACP) domains; and (d) one ketoreductase (KR) domain.
Genomic DNA clones (plasmids) encoding OrfA from both
Schizochytrium sp. ATCC 20888 and a daughter strain of ATCC 20888,
denoted Schizochytrium sp., strain N230D, have been isolated and
sequenced.
[0160] A genomic clone described herein as JK1126, isolated from
Schizochytrium sp. ATCC 20888, comprises, to the best of the
present inventors' knowledge, the nucleotide sequence spanning from
position 1 to 8730 of SEQ ID NO:1, and encodes the corresponding
amino acid sequence of SEQ ID NO:2. Genomic clone pJK1126 (denoted
pJK1126 OrfA genomic clone, in the form of an E. coli plasmid
vector containing "OrfA" gene from Schizochytrium ATCC 20888) was
deposited with the American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006,
and assigned ATCC Accession No. PTA-7648. The nucleotide sequence
of pJK1126 OrfA genomic clone, and the amino acid sequence encoded
by this plasmid are encompassed by the present invention.
[0161] Two genomic clones described herein as pJK306 OrfA genomic
clone and pJK320 OrfA genomic clone, isolated from Schizochytrium
sp. N230D, together (overlapping clones) comprise, to the best of
the present inventors' knowledge, the nucleotide sequence of SEQ ID
NO:1, and encode the amino acid sequence of SEQ ID NO:2. Genomic
clone pJK306 (denoted pJK306 OrfA genomic clone, in the form of an
E. coli plasmid containing 5' portion of OrfA gene from
Schizochytrium sp. N230D (2.2 kB overlap with pJK320)) was
deposited with the American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006,
and assigned ATCC Accession No. PTA-7641. The nucleotide sequence
of pJK306 OrfA genomic clone, and the amino acid sequence encoded
by this plasmid are encompassed by the present invention. Genomic
clone pJK320 (denoted pJK320 OrfA genomic clone, in the form of an
E. coli plasmid containing 3' portion of OrfA gene from
Schizochytrium sp. N230D (2.2 kB overlap with pJK306)) was
deposited with the American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, Va. 20110-2209 USA on Jun. 8, 2006,
and assigned ATCC Accession No. PTA-7644. The nucleotide sequence
of pJK320 OrfA genomic clone, and the amino acid sequence encoded
by this plasmid are encompassed by the present invention.
[0162] The first domain in OrfA is a KS domain, also referred to
herein as ORFA-KS, and the nucleotide sequence containing the
sequence encoding the ORFA-KS domain is represented herein as SEQ
ID NO:7 (positions 1-1500 of SEQ ID NO:1). The amino acid sequence
containing the ORFA-KS domain is represented herein as SEQ ID NO:8
(positions 1-500 of SEQ ID NO:2). It is noted that the ORFA-KS
domain contains an active site motif: DXAC* (*acyl binding site
C.sub.215). Also, a characteristic motif at the end of the
Schizochytrium KS region, GFGG, is present in this domain in SEQ ID
NO:2 and accordingly, in SEQ ID NO:8.
[0163] The second domain in OrfA is a MAT domain, also referred to
herein as ORFA-MAT, and the nucleotide sequence containing the
sequence encoding the ORFA-MAT domain is represented herein as SEQ
ID NO:9 (positions 1723-3000 of SEQ ID NO:1). The amino acid
sequence containing the ORFA-MAT domain is represented herein as
SEQ ID NO:10 (positions 575-1000 of SEQ ID NO:2). The MAT domain
comprises an aspartate at position 93 and a histidine at position
94 (corresponding to positions 667 and 668, respectively, of SEQ ID
NO:2). It is noted that the ORFA-MAT domain contains an active site
motif: GHS*XG (*acyl binding site S.sub.706), represented herein as
SEQ ID NO:11.
[0164] Domains 3-11 of OrfA are nine tandem ACP domains, also
referred to herein as ORFA-ACP (the first domain in the sequence is
ORFA-ACP1, the second domain is ORFA-ACP2, the third domain is
ORFA-ACP3, etc.). The first ACP domain, ORFA-ACP1, is contained
within the nucleotide sequence spanning from about position 3343 to
about position 3600 of SEQ ID NO:1 (OrfA). The nucleotide sequence
containing the sequence encoding the ORFA-ACP1 domain is
represented herein as SEQ ID NO:12 (positions 3343-3600 of SEQ ID
NO:1). The amino acid sequence containing the first ACP domain
spans from about position 1115 to about position 1200 of SEQ ID
NO:2. The amino acid sequence containing the ORFA-ACP1 domain is
represented herein as SEQ ID NO:13 (positions 1115-1200 of SEQ ID
NO:2). It is noted that the ORFA-ACP1 domain contains an active
site motif: LGIDS* (*pantetheine binding motif S.sub.1157),
represented herein by SEQ ID NO:14.
[0165] The nucleotide and amino acid sequences of all nine ACP
domains are highly conserved and therefore, the sequence for each
domain is not represented herein by an individual sequence
identifier. However, based on the information disclosed herein, one
of skill in the art can readily determine the sequence containing
each of the other eight ACP domains. All nine ACP domains together
span a region of OrfA of from about position 3283 to about position
6288 of SEQ ID NO:1, which corresponds to amino acid positions of
from about 1095 to about 2096 of SEQ ID NO:2. The nucleotide
sequence for the entire ACP region containing all nine domains is
represented herein as SEQ ID NO:16. The region represented by SEQ
ID NO:16 includes the linker segments between individual ACP
domains. The repeat interval for the nine domains is approximately
every 330 nucleotides of SEQ ID NO:16 (the actual number of amino
acids measured between adjacent active site serines ranges from 104
to 116 amino acids). Each of the nine ACP domains contains a
pantetheine binding motif LGIDS* (represented herein by SEQ ID
NO:14), wherein S* is the pantetheine binding site serine (S). The
pantetheine binding site serine (S) is located near the center of
each ACP domain sequence. At each end of the ACP domain region and
between each ACP domain is a region that is highly enriched for
proline (P) and alanine (A), which is believed to be a linker
region. For example, between ACP domains 1 and 2 is the sequence:
APAPVKAAAPAAPVASAPAPA, represented herein as SEQ ID NO: 15. The
locations of the active site serine residues (i.e., the pantetheine
binding site) for each of the nine ACP domains, with respect to the
amino acid sequence of SEQ ID NO:2, are as follows:
ACP1=S.sub.1157; ACP2=S.sub.1266; ACP3=S.sub.1377; ACP4=S.sub.1488;
ACP5=S.sub.1604; ACP6=S.sub.1715; ACP7=S.sub.1819; ACP8=S.sub.1930;
and ACP9=S.sub.2034. Given that the average size of an ACP domain
is about 85 amino acids, excluding the linker, and about 110 amino
acids including the linker, with the active site serine being
approximately in the center of the domain, one of skill in the art
can readily determine the positions of each of the nine ACP domains
in OrfA.
[0166] Domain 12 in OrfA is a KR domain, also referred to herein as
ORFA-KR, and the nucleotide sequence containing the sequence
encoding the ORFA-KR domain is represented herein as SEQ ID NO:17
(positions 6598-8730 of SEQ ID NO:1). The amino acid sequence
containing the ORFA-KR domain is represented herein as SEQ ID NO:18
(positions 2200-2910 of SEQ ID NO:2). Within the KR domain is a
core region with homology to short chain aldehyde-dehydrogenases
(KR is a member of this family). This core region spans from about
position 7198 to about position 7500 of SEQ ID NO:1, which
corresponds to amino acid positions 2400-2500 of SEQ ID NO:2.
Schizochytrium Open Reading Frame B (OrfB):
[0167] The complete nucleotide sequence for OrfB is represented
herein as SEQ ID NO:3. OrfB is a 6177 nucleotide sequence (not
including the stop codon) which encodes a 2059 amino acid sequence,
represented herein as SEQ ID NO:4. Within OrfB are four domains:
(a) one-keto acyl-ACP synthase (KS) domain; (b) one chain length
factor (CLF) domain; (c) one acyl transferase (AT) domain; and, (d)
one enoyl ACP-reductase (ER) domain.
[0168] Genomic DNA clones (plasmids) encoding OrfB from both
Schizochytrium sp. ATCC 20888 and a daughter strain of ATCC 20888,
denoted Schizochytrium sp., strain N230D, have been isolated and
sequenced.
[0169] A genomic clone described herein as pJK1129, isolated from
Schizochytrium sp. ATCC 20888, comprises, to the best of the
present inventors' knowledge, the nucleotide sequence of SEQ ID
NO:3, and encodes the amino acid sequence of SEQ ID NO:4. Genomic
clone pJK1129 (denoted pJK1129 OrfB genomic clone, in the form of
an E. coli plasmid vector containing "OrfB" gene from
Schizochytrium ATCC 20888) was deposited with the American Type
Culture Collection (ATCC), 10801 University Boulevard, Manassas,
Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No.
PTA-7649. The nucleotide sequence of pJK1126 OrfB genomic clone,
and the amino acid sequence encoded by this plasmid are encompassed
by the present invention.
[0170] A genomic clone described herein as pJK324 OrfB genomic
clone, isolated from Schizochytrium sp. N230D, comprises, to the
best of the present inventors' knowledge, the nucleotide sequence
of SEQ ID NO:3, and encodes the amino acid sequence of SEQ ID NO:4.
Genomic clone pJK324 (denoted pJK324 OrfB genomic clone, in the
form of an E. coli plasmid containing the OrfB gene sequence from
Schizochytrium sp. N230D) was deposited with the American Type
Culture Collection (ATCC), 10801 University Boulevard, Manassas,
Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No.
PTA-7643. The nucleotide sequence of pJK324 OrfB genomic clone, and
the amino acid sequence encoded by this plasmid are encompassed by
the present invention.
[0171] The first domain in OrfB is a KS domain, also referred to
herein as ORFB-KS, and the nucleotide sequence containing the
sequence encoding the ORFB-KS domain is represented herein as SEQ
ID NO:19 (positions 1-1350 of SEQ ID NO:3). The amino acid sequence
containing the ORFB-KS domain is represented herein as SEQ ID NO:20
(positions 1-450 of SEQ ID NO:4). This KS domain comprises a valine
at position 371 of SEQ ID NO:20 (also position 371 of SEQ ID
NO:20). It is noted that the ORFB-KS domain contains an active site
motif: DXAC* (*acyl binding site C.sub.196). Also, a characteristic
motif at the end of this KS region, GFGG, is present in this domain
in SEQ ID NO:4 and accordingly, in SEQ ID NO:20.
[0172] The second domain in OrfB is a CLF domain, also referred to
herein as ORFB-CLF, and the nucleotide sequence containing the
sequence encoding the ORFB-CLF domain is represented herein as SEQ
ID NO:21 (positions 1378-2700 of SEQ ID NO:3). The amino acid
sequence containing the ORFB-CLF domain is represented herein as
SEQ ID NO:22 (positions 460-900 of SEQ ID NO:4). It is noted that
the ORFB-CLF domain contains a KS active site motif without the
acyl-binding cysteine.
[0173] The third domain in OrfB is an AT domain, also referred to
herein as ORFB-AT, and the nucleotide sequence containing the
sequence encoding the ORFB-AT domain is represented herein as SEQ
ID NO:23 (positions 2701-4200 of SEQ ID NO:3). The amino acid
sequence containing the ORFB-AT domain is represented herein as SEQ
ID NO:24 (positions 901-1400 of SEQ ID NO:4). It is noted that the
ORFB-AT domain contains an active site motif of GxS*xG (*acyl
binding site S.sub.1140) that is characteristic of acyltransferse
(AT) proteins.
[0174] The fourth domain in OrfB is an ER domain, also referred to
herein as ORFB-ER, and the nucleotide sequence containing the
sequence encoding the ORFB-ER domain is represented herein as SEQ
ID NO:25 (positions 4648-6177 of SEQ ID NO:3). The amino acid
sequence containing the ORFB-ER domain is represented herein as SEQ
ID NO:26 (positions 1550-2059 of SEQ ID NO:4).
Schizochytrium Open Reading Frame C (OrfC):
[0175] The complete nucleotide sequence for OrfC is represented
herein as SEQ ID NO:5. OrfC is a 4506 nucleotide sequence (not
including the stop codon) which encodes a 1502 amino acid sequence,
represented herein as SEQ ID NO:6. Within OrfC are three domains:
(a) two FabA-like-hydroxy acyl-ACP dehydrase (DH) domains; and (b)
one enoyl ACP-reductase (ER) domain.
[0176] Genomic DNA clones (plasmids) encoding OrfC from both
Schizochytrium sp. ATCC 20888 and a daughter strain of ATCC 20888,
denoted Schizochytrium sp., strain N230D, have been isolated and
sequenced.
[0177] A genomic clone described herein as pJK1131, isolated from
Schizochytrium sp. ATCC 20888, comprises, to the best of the
present inventors' knowledge, the nucleotide sequence of SEQ ID
NO:5, and encodes the amino acid sequence of SEQ ID NO:6. Genomic
clone pJK1131 (denoted pJK1131 OrfC genomic clone, in the form of
an E. coli plasmid vector containing "OrfC" gene from
Schizochytrium ATCC 20888) was deposited with the American Type
Culture Collection (ATCC), 10801 University Boulevard, Manassas,
Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No.
PTA-7650. The nucleotide sequence of pJK1131 OrfC genomic clone,
and the amino acid sequence encoded by this plasmid are encompassed
by the present invention.
[0178] A genomic clone described herein as pBR002 OrfC genomic
clone, isolated from Schizochytrium sp. N230D, comprises, to the
best of the present inventors' knowledge, the nucleotide sequence
of SEQ ID NO:5, and encodes the amino acid sequence of SEQ ID NO:6.
Genomic clone pBR002 (denoted pBR002 OrfC genomic clone, in the
form of an E. coli plasmid vector containing the OrfC gene sequence
from Schizochytrium sp. N230D) was deposited with the American Type
Culture Collection (ATCC), 10801 University Boulevard, Manassas,
Va. 20110-2209 USA on Jun. 8, 2006, and assigned ATCC Accession No.
PTA-7642. The nucleotide sequence of pBR002 OrfC genomic clone, and
the amino acid sequence encoded by this plasmid are encompassed by
the present invention.
[0179] The first domain in OrfC is a DH domain, also referred to
herein as ORFC-DH1. This is one of two DH domains in OrfC, and
therefore is designated DH1. The nucleotide sequence containing the
sequence encoding the ORFC-DH1 domain is represented herein as SEQ
ID NO:27 (positions 1-1350 of SEQ ID NO:5). The amino acid sequence
containing the ORFC-DH1 domain is represented herein as SEQ ID
NO:28 (positions 1-450 of SEQ ID NO:6).
[0180] The second domain in OrfC is a DH domain, also referred to
herein as ORFC-DH2. This is the second of two DH domains in OrfC,
and therefore is designated DH2. The nucleotide sequence containing
the sequence encoding the ORFC-DH2 domain is represented herein as
SEQ ID NO:29 (positions 1351-2847 of SEQ ID NO:5). The amino acid
sequence containing the ORFC-DH2 domain is represented herein as
SEQ ID NO:30 (positions 451-949 of SEQ ID NO:6). This DH domain
comprises the amino acids 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 426-440 of SEQ ID NO:30.
[0181] The third domain in OrfC is an ER domain, also referred to
herein as ORFC-ER, and the nucleotide sequence containing the
sequence encoding the ORFC-ER domain is represented herein as SEQ
ID NO:31 (positions 2995-4506 of SEQ ID NO:5). The amino acid
sequence containing the ORFC-ER domain is represented herein as SEQ
ID NO:32 (positions 999-1502 of SEQ ID NO:6).
Thraustochytrium PUFA PKS System
[0182] In one embodiment, a Thraustochytrium PUFA PKS system
comprises at least the following biologically active domains: (a)
two enoyl-ACP reductase (ER) domain; (b) between five and ten or
more acyl carrier protein (ACP) domains, and in one aspect, eight
ACP domains; (c) two .beta.-ketoacyl-ACP synthase (KS) domains; (d)
one acyltransferase (AT) domain; (e) one .beta.-ketoacyl-ACP
reductase (KR) domain; (f) two FabA-like .beta.-hydroxyacyl-ACP
dehydrase (DH) domains; (g) one chain length factor (CLF) domain;
and (h) one malonyl-CoA:ACP acyltransferase (MAT) domain. In one
embodiment, a Thraustochytrium PUFA PKS system according to the
present invention also comprises at least one region or domain
containing a dehydratase (DH) conserved active site motif that is
not a part of a FabA-like DH domain. The structural and functional
characteristics of these domains are generally individually known
in the art (see, e.g., U.S. Patent Publication No. 2004035127,
supra).
[0183] There are three open reading frames that form the core
Thraustochytrium 23B PUFA PKS system described previously. The
domain structure of each open reading frame is as follows.
Thraustochytrium 23B Open Reading Frame A (OrfA):
[0184] The complete nucleotide sequence for Th. 23B OrfA is
represented herein as SEQ ID NO:38. Th. 23B OrfA is a 8433
nucleotide sequence (not including the stop codon) which encodes a
2811 amino acid sequence, represented herein as SEQ ID NO:39. SEQ
ID NO:38 encodes the following domains in Th. 23B OrfA: (a) one
.beta.-ketoacyl-ACP synthase (KS) domain; (b) one malonyl-CoA:ACP
acyltransferase (MAT) domain; (c) eight acyl carrier protein (ACP)
domains; and (d) one .beta.-ketoacyl-ACP reductase (KR) domain.
[0185] Two genomic clone described herein as Th23BOrfA_pBR812.1 and
Th23BOrfA_pBR811 (OrfA genomic clones), isolated from
Thraustochytrium 23B, together (overlapping clones) comprise, to
the best of the present inventors' knowledge, the nucleotide
sequence of SEQ ID NO:38, and encodes the amino acid sequence of
SEQ ID NO:39. Genomic clone Th23BOrfA_pBR812.1 (denoted
Th23BOrfA_pBR812.1 genomic clone, in the form of an E. coli plasmid
vector containing the OrfA gene sequence from Thraustochytrium 23B)
was deposited with the American Type Culture Collection (ATCC),
University Boulevard, Manassas, Va. 20110-2209 USA on Mar. 1, 2007,
and assigned ATCC Accession No. ______. The nucleotide sequence of
Th23BOrfA_pBR812.1, an OrfA genomic clone, and the amino acid
sequence encoded by this plasmid are encompassed by the present
invention. Genomic clone Th23BOrfA_pBR811 (denoted Th23BOrfA_pBR811
genomic clone, in the form of an E. coli plasmid vector containing
the OrfA gene sequence from Thraustochytrium 23B) was deposited
with the American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209 USA on Mar. 1, 2007, and
assigned ATCC Accession No. ______. The nucleotide sequence of
Th23BOrfA_pBR811, an OrfA genomic clone, and the amino acid
sequence encoded by this plasmid are encompassed by the present
invention.
[0186] The first domain in Th. 23B OrfA is a KS domain, also
referred to herein as Th. 23B OrfA-KS, and is contained within the
nucleotide sequence spanning from about position 1 to about
position 1500 of SEQ ID NO:38, represented herein as SEQ ID NO:40.
The amino acid sequence containing the Th. 23B KS domain is a
region of SEQ ID NO:39 spanning from about position 1 to about
position 500 of SEQ ID NO:39, represented herein as SEQ ID NO:41.
This region of SEQ ID NO:39 has a Pfam match to FabB
(.beta.-ketoacyl-ACP synthase) spanning from position 1 to about
position 450 of SEQ ID NO:39 (also positions 1 to about 450 of SEQ
ID NO:41). It is noted that the Th. 23B OrfA-KS domain contains an
active site motif: DXAC* (*acyl binding site C.sub.207). Also, a
characteristic motif at the end of the Th. 23B KS region, GFGG, is
present in positions 453-456 of SEQ ID NO:39 (also positions
453-456 of SEQ ID NO:41).
[0187] The second domain in Th. 23B OrfA is a MAT domain, also
referred to herein as Th. 23B OrfA-MAT, and is contained within the
nucleotide sequence spanning from between about position 1503 and
about position 3000 of SEQ ID NO:38, represented herein as SEQ ID
NO:42. The amino acid sequence containing the Th. 23B MAT domain is
a region of SEQ ID NO:39 spanning from about position 501 to about
position 1000, represented herein by SEQ ID NO:43. This region of
SEQ ID NO:39 has a Pfam match to FabD (malonyl-CoA:ACP
acyltransferase) spanning from about position 580 to about position
900 of SEQ ID NO:39 (positions 80-400 of SEQ ID NO:43). It is noted
that the Th. 23B OrfA-MAT domain contains an active site motif:
GHS*XG (*acyl binding site S.sub.697), represented by positions
695-699 of SEQ ID NO:39.
[0188] Domains 3-10 of Th. 23B OrfA are eight tandem ACP domains,
also referred to herein as Th. 23B OrfA-ACP (the first domain in
the sequence is OrfA-ACP1, the second domain is OrfA-ACP2, the
third domain is OrfA-ACP3, etc.). The first Th. 23B ACP domain, Th.
23B OrfA-ACP1, is contained within the nucleotide sequence spanning
from about position 3205 to about position 3555 of SEQ ID NO:38
(OrfA), represented herein as SEQ ID NO:44. The amino acid sequence
containing the first Th. 23B ACP domain is a region of SEQ ID NO:39
spanning from about position 1069 to about position 1185 of SEQ ID
NO:39, represented herein by SEQ ID NO:45.
[0189] The eight ACP domains in Th. 23B OrfA are adjacent to one
another and can be identified by the presence of the
phosphopantetheine binding site motif, LGXDS* (represented by SEQ
ID NO:46), wherein the S* is the phosphopantetheine attachment
site. The amino acid position of each of the eight S* sites, with
reference to SEQ ID NO:39, are 1128 (ACP1), 1244 (ACP2), 1360
(ACP3), 1476 (ACP4), 1592 (ACP5), 1708 (ACP6), 1824 (ACP7) and 1940
(ACP8). The nucleotide and amino acid sequences of all eight Th.
23B ACP domains are highly conserved and therefore, the sequence
for each domain is not represented herein by an individual sequence
identifier. However, based on the information disclosed herein, one
of skill in the art can readily determine the sequence containing
each of the other seven ACP domains in SEQ ID NO:38 and SEQ ID
NO:39.
[0190] All eight Th. 23B ACP domains together span a region of Th.
23B OrfA of from about position 3205 to about position 5994 of SEQ
ID NO:38, which corresponds to amino acid positions of from about
1069 to about 1998 of SEQ ID NO:39. The nucleotide sequence for the
entire ACP region containing all eight domains is represented
herein as SEQ ID NO:47. SEQ ID NO:47 encodes an amino acid sequence
represented herein by SEQ ID NO:48. SEQ ID NO:48 includes the
linker segments between individual ACP domains. The repeat interval
for the eight domains is approximately every 116 amino acids of SEQ
ID NO:48, and each domain can be considered to consist of about 116
amino acids centered on the active site motif (described
above).
[0191] The last domain in Th. 23B OrfA is a KR domain, also
referred to herein as Th. 23B OrfA-KR, which is contained within
the nucleotide sequence spanning from between about position 6001
to about position 8433 of SEQ ID NO:38, represented herein by SEQ
ID NO:49. The amino acid sequence containing the Th. 23B KR domain
is a region of SEQ ID NO:39 spanning from about position 2001 to
about position 2811 of SEQ ID NO:39, represented herein by SEQ ID
NO:50. This region of SEQ ID NO:39 has a Pfam match to FabG
(.beta.-ketoacyl-ACP reductase) spanning from about position 2300
to about 2550 of SEQ ID NO:39 (positions 300-550 of SEQ ID
NO:50).
Thraustochytrium. 23B Open Reading Frame B (OrfB):
[0192] The complete nucleotide sequence for Th. 23B OrfB is
represented herein as SEQ ID NO:51, which is a 5805 nucleotide
sequence (not including the stop codon) that encodes a 1935 amino
acid sequence, represented herein as SEQ ID NO:52. SEQ ID NO:51
encodes the following domains in Th. 23B OrfB: (a) one
.beta.-ketoacyl-ACP synthase (KS) domain; (b) one chain length
factor (CLF) domain; (c) one acyltransferase (AT) domain; and, (d)
one enoyl-ACP reductase (ER) domain.
[0193] A genomic clone described herein as Th23BOrfB_pBR800 (OrfB
genomic clone), isolated from Thraustochytrium 23B, comprises, to
the best of the present inventors' knowledge, the nucleotide
sequence of SEQ ID NO:51, and encodes the amino acid sequence of
SEQ ID NO:52. Genomic clone Th23BOrfB_pBR800 (denoted
Th23BOrfB_pBR800 genomic clone, in the form of an E. coli plasmid
vector containing the OrfB gene sequence from Thraustochytrium 23B)
was deposited with the American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, Va. 20110-2209 USA on Mar. 1,
2007, and assigned ATCC Accession No. ______. The nucleotide
sequence of Th23BOrfB_pBR800, an OrfB genomic clone, and the amino
acid sequence encoded by this plasmid are encompassed by the
present invention.
[0194] The first domain in the Th. 23B OrfB is a KS domain, also
referred to herein as Th. 23B OrfB-KS, which is contained within
the nucleotide sequence spanning from between about position 1 and
about position 1500 of SEQ ID NO:51 (Th. 23B OrfB), represented
herein as SEQ ID NO:53. The amino acid sequence containing the Th.
23B KS domain is a region of SEQ ID NO: 52 spanning from about
position 1 to about position 500 of SEQ ID NO:52, represented
herein as SEQ ID NO:54. This region of SEQ ID NO:52 has a Pfam
match to FabB (.beta.-ketoacyl-ACP synthase) spanning from about
position 1 to about position 450 (positions 1-450 of SEQ ID NO:54).
It is noted that the Th. 23B OrfB-KS domain contains an active site
motif: DXAC*, where C* is the site of acyl group attachment and
wherein the C* is at position 201 of SEQ ID NO:52. Also, a
characteristic motif at the end of the KS region, GFGG is present
in amino acid positions 434-437 of SEQ ID NO:52.
[0195] The second domain in Th. 23B OrfB is a CLF domain, also
referred to herein as Th. 23B OrfB-CLF, which is contained within
the nucleotide sequence spanning from between about position 1501
and about position 3000 of SEQ ID NO:51 (OrfB), represented herein
as SEQ ID NO:55. The amino acid sequence containing the CLF domain
is a region of SEQ ID NO: 52 spanning from about position 501 to
about position 1000 of SEQ ID NO:52, represented herein as SEQ ID
NO:56. This region of SEQ ID NO:52 has a Pfam match to FabB
(.beta.-ketoacyl-ACP synthase) spanning from about position 550 to
about position 910 (positions 50-410 of SEQ ID NO:56). Although CLF
has homology to KS proteins, it lacks an active site cysteine to
which the acyl group is attached in KS proteins.
[0196] The third domain in Th. 23B OrfB is an AT domain, also
referred to herein as Th. 23B OrfB-AT, which is contained within
the nucleotide sequence spanning from between about position 3001
and about position 4500 of SEQ ID NO:51 (Th. 23B OrfB), represented
herein as SEQ ID NO:58. The amino acid sequence containing the Th.
23B AT domain is a region of SEQ ID NO: 52 spanning from about
position 1001 to about position 1500 of SEQ ID NO:52, represented
herein as SEQ ID NO:58. This region of SEQ ID NO:52 has a Pfam
match to FabD (malonyl-CoA:ACP acyltransferase) spanning from about
position 1100 to about position 1375 (positions 100-375 of SEQ ID
NO:58). Although this AT domain of the PUFA synthases has homology
to MAT proteins, it lacks the extended motif of the MAT (key
arginine and glutamine residues) and it is not thought to be
involved in malonyl-CoA transfers. The GXS*XG motif of
acyltransferases is present, with the S* being the site of acyl
attachment and located at position 1123 with respect to SEQ ID
NO:52.
[0197] The fourth domain in Th. 23B OrfB is an ER domain, also
referred to herein as Th. 23B OrfB-ER, which is contained within
the nucleotide sequence spanning from between about position 4501
and about position 5805 of SEQ ID NO:51 (OrfB), represented herein
as SEQ ID NO:59. The amino acid sequence containing the Th. 23B ER
domain is a region of SEQ ID NO: 52 spanning from about position
1501 to about position 1935 of SEQ ID NO:52, represented herein as
SEQ ID NO:60. This region of SEQ ID NO:52 has a Pfam match to a
family of dioxygenases related to 2-nitropropane dioxygenases
spanning from about position 1501 to about position 1810 (positions
1-310 of SEQ ID NO:60). That this domain functions as an ER can be
further predicted due to homology to a newly characterized ER
enzyme from Streptococcus pneumoniae.
Thraustochytrium. 23B Open Reading Frame C (OrfC):
[0198] The complete nucleotide sequence for Th. 23B OrfC is
represented herein as SEQ ID NO:61, which is a 4410 nucleotide
sequence (not including the stop codon) that encodes a 1470 amino
acid sequence, represented herein as SEQ ID NO:62. SEQ ID NO:61
encodes the following domains in Th. 23B OrfC: (a) two FabA-like
.beta.-hydroxyacyl-ACP dehydrase (DH) domains, both with homology
to the FabA protein (an enzyme that catalyzes the synthesis of
trans-2-decenoyl-ACP and the reversible isomerization of this
product to cis-3-decenoyl-ACP); and (b) one enoyl-ACP reductase
(ER) domain with high homology to the ER domain of Schizochytrium
OrfB.
[0199] A genomic clone described herein as Th23BOrfC_pBR709A (OrfC
genomic clone), isolated from Thraustochytrium 23B, comprises, to
the best of the present inventors' knowledge, the nucleotide
sequence of SEQ ID NO:61, and encodes the amino acid sequence of
SEQ ID NO:62. Genomic clone Th23BOrfC_pBR709A (denoted
Th23BOrfC_pBR709A genomic clone, in the form of an E. coli plasmid
vector containing the OrfC gene sequence from Thraustochytrium 23B)
was deposited with the American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, Va. 20110-2209 USA on Mar. 1,
2007, and assigned ATCC Accession No. ______. The nucleotide
sequence of Th23BOrfC_pBR709A, an OrfC genomic clone, and the amino
acid sequence encoded by this plasmid are encompassed by the
present invention.
[0200] The first domain in Th. 23B OrfC is a DH domain, also
referred to herein as Th. 23B OrfC-DH1, which is contained within
the nucleotide sequence spanning from between about position 1 to
about position 1500 of SEQ ID NO:61 (OrfC), represented herein as
SEQ ID NO:63. The amino acid sequence containing the Th. 23B DH1
domain is a region of SEQ ID NO: 62 spanning from about position 1
to about position 500 of SEQ ID NO:62, represented herein as SEQ ID
NO:64. This region of SEQ ID NO:62 has a Pfam match to FabA, as
mentioned above, spanning from about position 275 to about position
400 (positions 275-400 of SEQ ID NO:64).
[0201] The second domain in Th. 23B OrfC is also a DH domain, also
referred to herein as Th. 23B OrfC-DH2, which is contained within
the nucleotide sequence spanning from between about position 1501
to about 3000 of SEQ ID NO:61 (OrfC), represented herein as SEQ ID
NO:65. The amino acid sequence containing the Th. 23B DH2 domain is
a region of SEQ ID NO: 62 spanning from about position 501 to about
position 1000 of SEQ ID NO:62, represented herein as SEQ ID NO:66.
This region of SEQ ID NO:62 has a Pfam match to FabA, as mentioned
above, spanning from about position 800 to about position 925
(positions 300-425 of SEQ ID NO:66).
[0202] The third domain in Th. 23B OrfC is an ER domain, also
referred to herein as Th. 23B OrfC-ER, which is contained within
the nucleotide sequence spanning from between about position 3001
to about position 4410 of SEQ ID NO:61 (OrfC), represented herein
as SEQ ID NO:67. The amino acid sequence containing the Th. 23B ER
domain is a region of SEQ ID NO: 62 spanning from about position
1001 to about position 1470 of SEQ ID NO:62, represented herein as
SEQ ID NO:68. This region of SEQ ID NO:62 has a Pfam match to the
dioxygenases related to 2-nitropropane dioxygenases, as mentioned
above, spanning from about position 1025 to about position 1320
(positions 25-320 of SEQ ID NO:68). This domain function as an ER
can also be predicted due to homology to a newly characterized ER
enzyme from Streptococcus pneumoniae.
Shewanella japonica PUFA PKS
[0203] There are five open reading frames that form the Shewanella
japonica core PUFA PKS system and its PPTase described previously.
The domain structure of each open reading frame is as follows.
[0204] SEQ ID NO:69 is the nucleotide sequence for Shewanella
japonica cosmid 3F3 and is found to contain 15 ORFs. The ORFs
related to the PUFA PKS system in this microorganism are
characterized as follows.
[0205] pfaA (nucleotides 10491-18854 of SEQ ID NO:69) encodes PFAS
A (SEQ ID NO:70), a PUFA PKS protein harboring the following
domains: .beta.-ketoacyl-synthase (KS) (nucleotides 10575-12029 of
SEQ ID NO:69, amino acids 29-513 of SEQ ID NO:70); malonyl-CoA: ACP
acyltransferase (MAT) (nucleotides 12366-13319 of SEQ ID NO:69,
amino acids 625-943 of SEQ ID NO:70); six tandem acyl-carrier
proteins (ACP) domains (nucleotides 14280-16157 of SEQ ID NO:69,
amino acids 1264-1889 of SEQ ID NO:70); .beta.-ketoacyl-ACP
reductase (KR) (nucleotides 17280-17684 of SEQ ID NO:69, amino
acids 2264-2398 of SEQ ID NO:70); and a region of the PFAS A
protein between amino acids 2399 and 2787 of SEQ ID NO:70
containing a dehydratase (DH) conserved active site motif
LxxHxxxGxxxxP (amino acids 2504-2516 of SEQ ID NO:70), referred to
herein as DH-motif region.
[0206] In PFAS A, a KS active site DXAC* is located at amino acids
226-229 of SEQ ID NO:70 with the C* being the site of the acyl
attachment. A MAT active site, GHS*XG, is located at amino acids
721-725 of SEQ ID NO:70, with the S* being the acyl binding site.
ACP active sites of LGXDS* are located at the following positions:
amino acids 1296-1300, amino acids 1402-1406, amino acids
1513-1517, amino acids 1614-1618, amino acids 1728-1732, and amino
acids 1843-1847 in SEQ ID NO:70, with the S* being the
phosphopantetheine attachment site. Between amino acids 2399 and
2787 of SEQ ID NO:70, the PFAS A also contains the dehydratase (DH)
conserved active site motif LxxHxxxGxxxxP (amino acids 2504-2516 of
SEQ ID NO:70) referenced above.
[0207] pfaB (nucleotides 18851-21130 of SEQ ID NO:69) encodes PFAS
B (SEQ ID NO:71), a PUFA PKS protein harboring the following
domain: acyltransferase (AT) (nucleotides 19982-20902 of SEQ ID
NO:69, amino acids 378-684 of SEQ ID NO:71).
[0208] In PFAS B, an active site GXS*XG motif is located at amino
acids 463-467 of SEQ ID NO:71, with the S* being the site of
acyl-attachment.
[0209] pfaC (nucleotides 21127-27186 of SEQ ID NO:69) encodes PFAS
C (SEQ ID NO:72), a PUFA PKS protein harboring the following
domains: KS (nucleotides 21139-22575 of SEQ ID NO:69, amino acids
5-483 of SEQ ID NO:72); chain length factor (CLF) (nucleotides
22591-23439 of SEQ ID NO:69, amino acids 489-771 of SEQ ID NO:72);
and two FabA 3-hydroxyacyl-ACP dehydratases, referred to as DH1
(nucleotides 25408-25836 of SEQ ID NO:69, amino acids 1428-1570 of
SEQ ID NO:72) and DH2 (nucleotides 26767-27183 of SEQ ID NO:69,
amino acids 1881-2019 of SEQ ID NO:72).
[0210] In PFAS C, a KS active site DXAC* is located at amino acids
211-214 of SEQ ID NO:72 with the C* being the site of the acyl
attachment.
[0211] pfaD (nucleotides 27197-28825 of SEQ ID NO:69) encodes the
PFAS D (SEQ ID NO:73), a PUFA PKS protein harboring the following
domain: an enoyl reductase (ER) (nucleotides 27446-28687 of SEQ ID
NO:69, amino acids 84-497 of SEQ ID NO:73).
[0212] pfaE (nucleotides 6150-7061 of SEQ ID NO:69 on the reverse
complementary strand) encodes PFAS E (SEQ ID NO:74), a
4'-phosphopantetheinyl transferase (PPTase) with the identified
domain (nucleotides 6504-6944 of SEQ ID NO:69, amino acids 40-186
of SEQ ID NO:74).
Shewanella olleyana PUFA PKS
[0213] There are five open reading frames that form the Shewanella
olleyana core PUFA PKS system and its PPTase described previously.
The domain structure of each open reading frame is as follows.
[0214] SEQ ID NO:75 is the nucleotide sequence for Shewanella
olleyana cosmid 9A10 and was found to contain 17 ORFs. The ORFs
related to the PUFA PKS system in this microorganism are
characterized as follows.
[0215] pfaA (nucleotides 17437-25743 of SEQ ID NO:75) encodes PFAS
A (SEQ ID NO:76), a PUFA PKS protein harboring the following
domains: .beta.-ketoacyl-synthase (KS) (nucleotides 17521-18975 of
SEQ ID NO:75, amino acids 29-513 of SEQ ID NO:76); malonyl-CoA: ACP
acyltransferase (MAT) (nucleotides 19309-20265 of SEQ ID NO:75,
amino acids 625-943 of SEQ ID NO:76); six tandem acyl-carrier
proteins (ACP) domains (nucleotides 21259-23052 of SEQ ID NO:75,
amino acids 1275-1872 of SEQ ID NO:76); .beta.-ketoacyl-ACP
reductase (KR) (nucleotides 24154-24558 of SEQ ID NO:75, amino
acids 2240-2374 of SEQ ID NO:76); and a region of the PFAS A
protein between amino acids 2241 and 2768 of SEQ ID NO:76
containing a dehydratase (DH) conserved active site motif
LxxHxxxGxxxxP (amino acids 2480-2492 of SEQ ID NO:76), referred to
herein as DH-motif region.
[0216] In PFAS A, a KS active site DXAC* is located at AA 226-229
of SEQ ID NO:76 with the C* being the site of the acyl attachment.
A MAT active site, GHS*XG, is located at amino acids 721-725 of SEQ
ID NO:76 with the S* being the acyl binding site. ACP active sites
of LGXDS* are located at: amino acids 1307-1311, amino acids
1408-1412, amino acids 1509-1513, amino acids 1617-1621, amino
acids 1721-1725, and amino acids 1826-1830 in SEQ ID NO:76, with
the S* being the phosphopantetheine attachment site. Between amino
acids 2241 and 2768 of SEQ ID NO:76, the PFAS A also contains the
dehydratase (DH) conserved active site motif LxxHxxxGxxxxP (amino
acids 2480-2492 of SEQ ID NO:76) referenced above.
[0217] pfaB (nucleotides 25740-27971 of SEQ ID NO:75) encodes PFAS
B (SEQ ID NO:77), a PUFA PKS protein harboring the following
domain: acyltransferase (AT) (nucleotides 26837-27848 of SEQ ID
NO:75, amino acids 366-703 of SEQ ID NO:77).
[0218] In PFAS B, an active site GXS*XG motif is located at amino
acids 451-455 of SEQ ID NO:77 with the S* being the site of
acyl-attachment.
[0219] pfaC (nucleotides 27968-34030 of SEQ ID NO:75) encodes PFAS
C (SEQ ID NO:78), a PUFA PKS protein harboring the following
domains: KS (nucleotides 27995-29431 SEQ ID NO:75, amino acids
10-488 SEQ ID NO:78); chain length factor (CLF) (nucleotides
29471-30217 SEQ ID NO:75, amino acids 502-750 SEQ ID NO:78); and
two FabA 3-hydroxyacyl-ACP dehydratases, referred to as DH1
(nucleotides 32258-32686 SEQ ID NO:75, amino acids 1431-1573 SEQ ID
NO:78), and DH2 (nucleotides 33611-34027 of SEQ ID NO:75, amino
acids 1882-2020 of SEQ ID NO:78).
[0220] In PFAS C, a KS active site DXAC* is located at amino acids
216-219 of SEQ ID NO:78 with the C* being the site of the acyl
attachment.
[0221] pfaD (nucleotides 34041-35669 of SEQ ID NO:75) encodes the
PFAS D (SEQ ID NO:79), a PUFA PKS protein harboring the following
domain: an enoyl reductase (ER) (nucleotides 34290-35531 of SEQ ID
NO:75, amino acids 84-497 of SEQ ID NO:79).
[0222] pfaE (nucleotides 13027-13899 of SEQ ID NO:75 on the reverse
complementary strand) encodes PFAS E (SEQ ID NO:80), a
4'-phosphopantetheinyl transferase (PPTase) with the identified
domain (nucleotides 13369-13815 of SEQ ID NO:75, amino acid 29-177
of SEQ ID NO:80).
Other PUFA PKS Sequences, Including Optimized PUFA PKS
Sequences
[0223] The invention includes various optimized sequences for use
in the expression of PUFA PKS systems in heterologous hosts,
examples of which are provided below. One of skill in the art will
be able to produce optimized sequences, in particular, sequences
optimized for a preferred codon usage or better expression and
function in a heterologous host.
sOrf A
[0224] SEQ ID NO:35, denoted sOrfA, represents the nucleic acid
sequence encoding OrfA from Schizochytrium (SEQ ID NO:1) that has
been resynthesized for optimized codon usage in yeast. SEQ ID NO:1
and SEQ ID NO:35 each encode SEQ ID NO:2.
sOrfB
[0225] SEQ ID NO:36, denoted sOrfB, represents the nucleic acid
sequence encoding OrfB from Schizochytrium (SEQ ID NO:3) that has
been resynthesized for optimized codon usage in yeast. SEQ ID NO:3
and SEQ ID NO:36 each encode SEQ ID NO:4.
[0226] SEQ ID NO:37, denoted OrfB*, represents a nucleic acid
sequence encoding OrfB from Schizochytrium (SEQ ID NO:3) that has
been resynthesized within a portion of SEQ ID NO:3 for use in plant
cells, and that was derived from a very similar sequence initially
developed for optimized codon usage in E. coli, also referred to as
OrfB*. OrfB* in both forms (for E. coli and for plants) is
identical to SEQ ID NO:3 with the exception of a resynthesized
BspHI (nucleotide 4415 of SEQ ID NO:3) to a SacII fragment (unique
site in SEQ ID NO:3). Both versions (E. coli and plant) have two
other codon modifications near the start of the gene as compared
with the original genomic sequence of orfB (SEQ ID NO:3). First,
the fourth codon, arginine (R), was changed from CGG in the genomic
sequence to CGC in orfB*. Second, the fifth codon, asparagine (N),
was changed from AAT in the genomic sequence to AAC in orf B*. In
order to facilitate cloning of this gene into the plant vectors to
create SEQ ID NO:37, a PstI site (CTGCAG) was also engineered into
the E. coli orfB* sequence 20 bases from the start of the gene.
This change did not alter the amino acid sequence of the encoded
protein. Both SEQ ID NO:37 and SEQ ID NO:3 (as well as the OrfB*
form for E. coli) encode SEQ ID NO:4.
Accessory Proteins and Additional Targets and Strategies for
Improved PUFA Production and Accumulation
[0227] According to the present invention, a PUFA PKS system for
production and/or accumulation of PUFAs in a heterologous host or
improved production and/or accumulation of PUFAs in an endogenous
host, the PUFA PKS system preferably makes use of one or more of
the various targets or strategies described above for the
production of PUFAs (see the six guidelines and strategies
described above). These strategies include, among other things, the
use of various accessory proteins, which are defined herein as
proteins that are not considered to be part of the core PUFA PKS
system as described above (i.e., not part of the PUFA synthase
enzyme complex itself), but which may be, or are, necessary for
PUFA production or at least for efficient PUFA production using the
core PUFA synthase enzyme complex of the present invention. These
strategies also include various genetic modifications to increase
the flux of substrate, malonyl CoA, through the PUFA synthase
pathway by enhancing its ability to compete for the malonyl-CoA
pool(s). Variations of these embodiments of the invention are
described below.
Phosphopantetheinyl Transferase (PPTase)
[0228] As discussed under the general guidelines and strategies for
the production of PUFAs in a heterologous host above, in order to
produce PUFAs, a PUFA PKS system must work with an accessory
protein that transfers a 4'-phosphopantetheinyl moiety from
coenzyme A to the acyl carrier protein (ACP) domain(s). Therefore,
a PUFA PKS system can be considered to include at least one
4'-phosphopantetheinyl transferase (PPTase) domain, or such a
domain can be considered to be an accessory domain or protein to
the PUFA PKS system. Structural and functional characteristics of
PPTases have been described in detail, for example, in U.S. Patent
Application Publication No. 20020194641; U.S. Patent Application
Publication No. 20040235127; and U.S. Patent Application
Publication No. 20050100995.
[0229] According to the present invention, a domain or protein
having 4'-phosphopantetheinyl transferase (PPTase) biological
activity (function) is characterized as the enzyme that transfers a
4'-phosphopantetheinyl moiety from Coenzyme A to the acyl carrier
protein (ACP). This transfer to an invariant serine reside of the
ACP activates the inactive apo-form to the holo-form. In both
polyketide and fatty acid synthesis, the phosphopantetheine group
forms thioesters with the growing acyl chains. The PPTases are a
family of enzymes that have been well characterized in fatty acid
synthesis, polyketide synthesis, and non-ribosomal peptide
synthesis. The sequences of many PPTases are known, and crystal
structures have been determined (e.g., Reuter K, Mofid M R,
Marahiel M A, Ficner R. "Crystal structure of the surfactin
synthetase-activating enzyme sfp: a prototype of the
4'-phosphopantetheinyl transferase superfamily" EMBO J. 1999 Dec.
1; 18(23):6823-31) as well as mutational analysis of amino acid
residues important for activity (Mofid M R, Finking R, Essen L O,
Marahiel M A. "Structure-based mutational analysis of the
4'-phosphopantetheinyl transferases Sfp from Bacillus subtilis:
carrier protein recognition and reaction mechanism" Biochemistry.
2004 Apr. 13; 43(14):4128-36). These invariant and highly conserved
amino acids in PPTases are contained within the pfaE ORFs from both
Shewanella strains described above.
[0230] One heterologous PPTase which has been demonstrated
previously to recognize the OrfA ACP domains described herein as
substrates is the Het I protein of Nostoc sp. PCC 7120 (formerly
called Anabaena sp. PCC 7120). Het I is present in a cluster of
genes in Nostoc known to be responsible for the synthesis of long
chain hydroxy-fatty acids that are a component of a glyco-lipid
layer present in heterocysts of that organism (Black and Wolk,
1994, J. Bacteriol. 176, 2282-2292; Campbell et al., 1997, Arch.
Microbiol. 167, 251-258). Het I is likely to activate the ACP
domains of a protein, Hgl E, present in that cluster. The two ACP
domains of Hgl E have a high degree of sequence homology to the ACP
domains found in Schizochytrium Orf A. SEQ ID NO:34 represents the
amino acid sequence of the Nostoc Het I protein, and is a
functional PPTase that can be used with a PUFA PKS system described
herein, including the PUFA PKS systems from Schizochytrium and
Thraustochytrium. SEQ ID NO:34 is encoded by SEQ ID NO:33. The
endogenous start codon of Het I has not been identified (there is
no methionine present in the putative protein). There are several
potential alternative start codons (e.g., TTG and ATT) near the 5'
end of the open reading frame. No methionine codons (ATG) are
present in the sequence. However, the construction of a Het I
expression construct was completed using PCR to replace the
furthest 5' potential alternative start codon (TTG) with a
methionine codon (ATG, as part of an NdeI restriction enzyme
recognition site), and introducing an XhoI site at the 3' end of
the coding sequence, and the encoded PPTase (SEQ ID NO:34) has been
shown to be functional.
[0231] Another heterologous PPTase which has been demonstrated
previously to recognize the OrfA ACP domains described herein as
substrates is sfp, derived from Bacillus subtilis. Sfp has been
well characterized, and is widely used due to its ability to
recognize a broad range of substrates. Based on published sequence
information (Nakana, et al., 1992, Molecular and General Genetics
232: 313-321), an expression vector was previously produced for sfp
by cloning the coding region, along with defined up- and downstream
flanking DNA sequences, into a pACYC-184 cloning vector. This
construct encodes a functional PPTase as demonstrated by its
ability to be co-expressed with Schizochytrium Orfs A, B*, and C in
E. coli which, under appropriate conditions, resulted in the
accumulation of DHA in those cells (see U.S. Patent Application
Publication No. 20040235127).
[0232] When genetically modifying organisms (e.g., microorganisms
or plants) to express a PUFA PKS system according to the present
invention, some host organisms may endogenously express accessory
proteins that are needed to work with the PUFA PKS to produce PUFAs
(e.g., PPTases). However, some organisms may be transformed with
nucleic acid molecules encoding one or more accessory proteins
described herein to enable and/or to enhance production of PUFAs by
the organism, even if the organism endogenously produces a
homologous accessory protein (i.e., some heterologous accessory
proteins may operate more effectively or efficiently with the
transformed PUFA synthase proteins than the host cells' endogenous
accessory protein). The present invention provides an example of
bacteria, yeast and plants that have been genetically modified with
the PUFA PKS system of the present invention that includes an
accessory PPTase.
[0233] Accordingly, one embodiment of the invention relates to a
genetically modified host cell or organism (e.g., a microorganism
or a plant, or cells thereof), wherein the host cell or organism
has been genetically modified to express a core PUFA PKS system as
described herein, and also a PPTase as described herein. Suitable
PPTases are described above and are also described in the art. The
PPTase may be expressed on the same or a different construct as one
or more of the nucleic acid molecules encoding the core PUFA PKS
protein or proteins. Both embodiments are illustrated in the
Examples (see Examples 12 and 13). In one aspect, the PPTase is the
Nostoc HetI (represented herein by SEQ ID NOs:33 and 34).
[0234] In one embodiment of the invention, PUFA production and
accumulation is enhanced by reducing (inhibiting, downregulating,
decreasing) the expression or activity of an endogenous PPTase
expressed by a host cell or host organism (e.g., to avoid
competition with the PPTase introduced with the PUFA PKS enzymes
according to this embodiment). Inhibition of endogenous PPTase
activity can be achieved by any suitable method of deletion or
inactivation of genes, including, but not limited to, use of
antisense RNA, RNAi, co-suppression, or introduction of
mutations).
[0235] The invention includes the expression of exogenous PPTases
(alone or in combination with inhibition of endogenous PPTases) in
conjunction with expression of a PUFA synthase as described herein,
which are utilized alone or in combination with any one or more
strategies described herein (e.g., any one, two, three, four or
five of: codon optimization, organelle-targeting, enhancement of
PUFA synthase competition for malonyl CoA (e.g., by inhibition of
FAS), expression of an acyl CoA synthetase, and/or expression of
one or more acyltransferases or related enzymes), to increase PUFA
production and/or accumulation in a heterologous host.
Modification of Malonyl CoA Flux/Inhibition of FAS
[0236] As discussed above, the substrate for the PUFA PKS system
(PUFA synthase), malonyl-CoA, is also used by fatty acid synthase
systems (FASs), cytoplasmic fatty acid elongation reactions and
other enzymes (e.g., chalcone synthase). Therefore, the PUFA
synthase competes with these other enzyme systems for the
malonyl-CoA. Accordingly, one embodiment of the invention relates
to methods and genetic modifications to increase the flux of
malonyl CoA through the PUFA synthase pathway by enhancing the
ability of PUFA synthase enzymes to compete for the malonyl-CoA
pool(s). Methods proposed herein include, but are not limited to,
1) inhibition of competing pathways, including inhibition of any
elements in the FAS pathway, e.g., by reducing expression levels of
enzymes or subunits involved in those pathways (e.g., by use of
antisense RNA, RNAi, co-suppression, or mutations), 2) expression
of the PUFA synthase in heterologous hosts in which competing
pathways have been reduced or blocked (e.g., in Canola where the
ability to elongate fatty acids in the cytoplasm has been blocked),
and/or 3) by increasing the pool of malonyl-CoA (e.g., by
expression of acetyl-CoA carboxylase).
[0237] More specifically, in one aspect, the present invention also
includes the genetic modification of host organisms that produce
PUFAs, and particularly host organisms that express a heterologous
PUFA PKS system, to delete or inactivate gene(s), or to reduce the
level of activity of enzymes encoded by those genes, that may
compete with or interfere with PUFA production and/or accumulation
by the PUFA PKS system. For example, the present inventors have
found that by reducing the FAS activity in a host organism that has
been transformed with a PUFA PKS system, PUFA production and
accumulation improves as compared to host organisms that retain the
normal level of FAS activity (see exemplary experiments in
Schizochytrium, as well as experiments detailed for yeast and
plants in the Examples).
[0238] In one embodiment, various enzymes that inhibit the
production of fatty acids through the FAS pathway is envisioned.
Many enzymes can be suitable targets for this embodiment of the
invention, and two particularly useful targets are exemplified and
described in detail below. The inventors have demonstrated the
ability to knock out an FAS enzyme in Schizochytrium (see
Examples), and this strategy can be applied to heterologous hosts.
In another embodiment, the inventors have demonstrated the ability
to inhibit the FAS system by biochemical methods in a yeast host,
resulting in improved PUFA production in yeast expressing a PUFA
synthase and a PPTase, as compared to in the absence of the
biochemical targeting of the FAS system. Certain other hosts may be
amenable to similar strategies.
[0239] Finally, in plants, the present inventors have demonstrated
that inhibition of the FAS pathway by inhibition of KasII or KasIII
using antisense or RNAi technology improves PUFA production in
heterologous hosts expressing a PUFA synthase and a PPTase. While
the invention is not limited to these particular targets, it is one
aspect of the invention to target one or both of these enzymes for
inhibition in conjunction with expression of a PUFA synthase and
PPTase as described herein, alone or in combination with other
strategies described herein (e.g., codon optimization,
organelle-targeting, expression of an acyl CoA synthetase, and/or
expression of one or more acyltransferases or related enzymes), to
increase PUFA production and/or accumulation in a heterologous
host.
[0240] In seeds the lipids, mainly in the form of triacylglycerols
(TAGs), are derived from assimilates through an elaborate enzymatic
pathway. Generally, reduced carbon is delivered to the seed via the
phloem from other parts of the plant. In plant seeds the
biosynthesis of TAGs is carried out intracellularly within
different organelles (Ohrolgge and Browse, 1995, Plant Cell 7:
957-970). Within the plastids, short carbon precursors are
converted to long chain fatty acids by the Type II soluble fatty
acid synthase (FAS) complex (Slabas and Fawcett, 1992, Plant
Molecular Biology 19: 169-191), which reiteratively adds C2-units
to a fatty acyl chain and prepares the chain for the next round of
elongation. The condensation of eight or nine rounds of C2-units
yields the C16 and C18 fatty acids that characterize membrane
lipids. The initial FAS activity is performed by the nuclear
encoded, plastid targeted enzyme malonyl-CoA:ACP transacylase
(MCAT), which transfers the malonyl group from malonyl-CoA to acyl
carrier protein (ACP) (Yasuno et al., 2004, Journal of Biological
Chemistry 292: 8242-8251). This forms the substrate, malonyl-ACP,
which provides the C2-units for subsequent elongation. The next
step in the synthesis is achieved through the catalytic activity of
the nuclear encoded, plastid targeted .beta.-ketoacyl-acyl carrier
protein synthetase III (KAS III), in which the condensation of
malonyl-CoA to the donor, malonyl-ACP, results in butyryl (C4)-ACP.
All subsequent extensions of the ACP-activated acyl chains is
carried out by the nuclear encoded, plastid targeted
3-ketoacyl-acyl carrier protein synthetase I (KAS I) and
.beta.-ketoacyl-acyl carrier protein synthetase II (KAS II)
isozymes. KAS I catalyzes the condensation reactions converting
C4-ACP to C16-ACP by utilizing butyryl (C4)- to myristoyl
(C14)-ACPs as substrates, and KAS II is performs the last step to
yield stearoyl (C18)-ACP by utilizing palmitoyl (C16)-ACP (Carlsson
et al., 2002, Plant Journal 29: 761-770). Therefore, by inhibiting
or attenuating the expression of KasIII or KasII, inhibition of
fatty acid biosynthesis during seed development may be
achieved.
[0241] In one embodiment, the invention includes the transformation
of a heterologous host organism or cell with a nucleic acid
molecule comprising RNAi targeting either of KasII or KasIII in the
host cell. In one embodiment, the host cell is a plant cell. In one
embodiment, the invention includes the transformation of a
heterologous host organism or cell with a nucleic acid molecule
comprising antisense targeting either of KasII or KasIII in the
host cell. In a preferred embodiment, the host cell is a plant
cell.
[0242] In one embodiment, the invention includes transformation of
a heterologous host organism or cell with a nucleic acid molecule
comprising the nucleic acid sequence represented by SEQ ID NO:122,
which is KAS II RNAi with CHSA intron as described in Example 13.
In one embodiment, the invention includes transformation of a
heterologous host organism or cell with a nucleic acid molecule
comprising the nucleic acid sequence represented by SEQ ID NO:124,
which is KAS III RNAi with CHSA intron as described in Example 13.
In one embodiment, the invention includes transformation of a
heterologous host organism or cell with a nucleic acid molecule
comprising the nucleic acid sequence represented by SEQ ID NO:123,
which is KAS II antisense nucleic acid sequence as described in
Example 13. In one embodiment, the invention includes
transformation of a heterologous host organism or cell with a
nucleic acid molecule comprising the nucleic acid sequence
represented by SEQ ID NO:125, which is KAS III antisense nucleic
acid sequence as described in Example 13.
[0243] Additional methods for enhancing the ability of PUFA
synthase enzymes to compete for the malonyl-CoA pool(s) include
expression of the PUFA synthase in heterologous hosts in which
competing pathways have been reduced or blocked (e.g., in Canola
where the ability to elongate fatty acids in the cytoplasm has been
blocked). Other suitable heterologous hosts can be selected
(naturally occurring organisms and/or mutants identified by
selection, random mutation and screening, and/or directed mutation)
by techniques such as tilling, breeding, marker assisted selection,
etc., for reduced or blocked competing pathways, such as FAS
pathways and the like.
[0244] Expression of other enzymes, such as acetyl-CoA carboxylase,
may also increase the malonyl CoA pool available for all enzyme
systems, and thus improve flux through the PUFA PKS system.
[0245] The invention includes the enactment of any of the
embodiments for improving the ability of a PUFA PKS system to use
malonyl CoA with the expression of exogenous PPTases (alone or in
combination with inhibition of endogenous PPTases) in conjunction
with expression of a PUFA synthase as described herein, which are
utilized alone or in combination with any one or more strategies
described herein (e.g., any one, two, three, or four of: codon
optimization, organelle-targeting, expression of an acyl CoA
synthetase, and/or expression of one or more acyltransferases or
related enzymes), to increase PUFA production and/or accumulation
in a heterologous host.
Acyl-CoA Synthetase
[0246] Another embodiment of the present invention provides
acyl-CoA synthetase (ACoAS) proteins that catalyze the conversion
of long chain PUFA free fatty acids (FFA) to acyl-CoA.
[0247] The present inventors have determined that an endogenous
producer of PUFAs by the PUFA PKS system, Schizochytrium, possesses
one or more ACoASs that may be capable of converting the FFA
products of its PUFA PKS system into acyl-CoA. This is evident by
the fact that high levels of PUFAs accumulate in those fractions in
this organism. Therefore, Schizochytrium, as well as other
organisms that endogenously contain a PUFA PKS system (e.g., other
Thraustochytrids) or other eukaryotes that produce PUFAs (such as
Thalassiosira pseudonana or Crypthecodinium cohnii), represent
excellent sources for genes encoding enzymes that are useful in
permitting or increasing the accumulation of the products of a PUFA
PKS system expressed in a heterologous host.
[0248] The present inventors have identified in Schizochytrium nine
nucleic acid sequences encoding proteins with homology to proteins
with known or suspected acyl-CoA synthetase (ACoAS) activity. The
present inventors believe that one or several of these sequences is
associated with a gene encoding an ACoAS capable of converting the
FFA products of the Schizochytrium PUFA synthase into acyl-CoA, and
have demonstrated the ability to use several of these sequences to
increase PUFA production and/or accumulation in a host organism. As
such they will have great utility for increasing the accumulation
of PUFAs in the heterologous host into which the Schizochytrium
PUFA synthase or another PUFA synthase is expressed. Without being
bound by theory, the present inventors believe that the ACoAS
discovered by the present inventors are useful for increasing PUFA
accumulation in hosts expressing a PUFA synthase with a product
profile similar to that of Schizochytrium, as well as in hosts
expressing a PUFA synthase with a product profile that is different
than that of the Schizochytrium PUFA synthase. Indeed, the Examples
presented herein demonstrate that several ACoASs from
Schizochytrium increase the accumulation of PUFAs in yeast strains
that have been genetically modified with a Schizochytrium PUFA PKS
system and also in plants that have been similarly genetically
modified. In addition, the Schizochytrium ACoASs are expected to be
effective in recognizing the EPA produced by PUFA synthases from
other organisms if that EPA is present as a FFA. Moreover, given
the disclosure provided by the present invention, the genes
encoding ACoASs from other organisms can be identified and obtained
for use in heterologous host organisms expressing those PUFA
synthases. Each of these ACoAS proteins and the nucleic acids
encoding the same are encompassed by the present invention, as well
as homologues and biologically active fragments thereof. These
proteins and nucleic acid molecules will be discussed in detail
below and in the Examples.
[0249] One embodiment of the present invention relates to an
isolated acyl-CoA synthetase (ACoAS) that catalyzes the conversion
of long chain PUFA free fatty acids (FFA) to acyl-CoA. In one
aspect of the invention, the isolated ACoAS is derived from an
organism that endogenously expresses a PUFA PKS system (PUFA
synthase). Such organisms include, but are not limited to, a
Thraustochytrid. In one aspect, the isolated ACoAS is derived from
Schizochytrium, Thraustochytrium, or Ulkenia. In another aspect,
the isolated ACoAS is derived from Schizochytrium ATCC 20888 or
from Schizochytrium sp. strain N230D, which is a strain derived
from Schizochytrium ATCC 20888 by mutagenesis and selection for
improved oil production. In another aspect, any ACoAS that
functions in conjunction with any PUFA PKS system to increase the
production and/or accumulation of PUFAs in a host cell or organism
can be used in the present invention. The invention is not limited
to those specific examples described herein.
[0250] In another aspect, the isolated ACoAS is encoded by a
nucleotide sequence selected from any one of SEQ ID NOs:82, 84, 86,
88, 90, 92, 94, 96, or 98. In another aspect, the isolated ACoAS is
encoded by a degenerate nucleic acid sequence encoding a protein
that is encoded by a nucleotide sequence selected from any one of
SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, or 98. In yet another
aspect, the isolated ACoAS comprises an amino acid sequence
selected from any one of SEQ ID NOs:83, 85, 87, 89, 91, 93, 95, 97
or 99, or a homologue of any of such amino acid sequences
(described below), including any biologically active fragments or
domains of such sequences. In a preferred embodiment, the isolated
ACoAS comprises an amino acid sequence represented herein by SEQ ID
NO: 83, 85, 87, 89, 91, 93, 95, 97 or 99, or a homologue of such
amino acid sequence. In a more preferred embodiment, the isolated
ACoAS comprises an amino acid sequence represented herein by SEQ ID
NO:83, 85, 87, 91 or 97, or a homologue of such sequence, with SEQ
ID NO:83, 85, or 97 being particularly preferred. Combinations of
any one or more acyl-CoA synthetases are also encompassed by the
invention.
[0251] The invention includes the expression of one or more
acyl-CoA synthetases as described and exemplified herein with a
PUFA synthase as described herein and with an exogenous PPTase
(alone or in combination with inhibition of endogenous PPTases),
which are utilized alone or in combination with any one or more
strategies described herein (e.g., any one, two, three, or four of:
codon optimization, organelle-targeting, enhancement of PUFA
synthase competition for malonyl CoA (e.g., by inhibition of FAS),
and/or expression of one or more acyltransferases or related
enzymes), to increase PUFA production and/or accumulation in a
heterologous host.
Acyltransferases
[0252] Relating to another strategy for increasing production
and/or accumulation of PUFAs in a heterologous host described
above, another embodiment of the present invention provides
additional acyltransferase proteins that utilize PUFA-CoA as
substrates in forming PL or TAG (e.g., 3-glycerol-phosphate
acyltransferases (GPAT), lysophosphatidic acid acyltransferases
(LPAAT) and diacylglycerol acyltransferases (DAGAT)) or other
acyltransferases that may result in enrichment of PUFAs in PL or
TAG (e.g., phospholipid:diacylglycerol acyltransferases (PDAT)).
The present invention includes such isolated proteins and
homologues thereof, nucleic acid molecules encoding such proteins,
genetically modified organisms expressing such proteins, and
various methods of using such proteins, particularly to enhance
PUFA production and accumulation in an organism.
[0253] In addition, the present inventors also disclose herein that
enzymes that can utilize PUFA-CoA as substrates in forming PL or
TAG, and therefore represent additional accessory proteins that can
be used in heterologous host organisms expressing PUFA synthases to
enhance the accumulation of PUFAs produced by the PUFA synthases.
Candidate enzymes include, but are not limited to,
3-glycerol-phosphate acyltransferases (GPAT), lysophosphatidic acid
acyltransferases (LPAAT) and diacylglycerol acyltransferases
(DAGAT). Each of these acyl-CoA-utilizing proteins and the nucleic
acids encoding the same are encompassed by the present invention.
For example, a Schizochytrium nucleic acid sequence has been
identified that is believed to encode an enzyme possessing DAGAT
activity (see e.g., ScDAGAT). In addition, Crypthecodinium cohnii
sequences have been identified that are believed to encode enzymes
possessing LPAAT or DAGAT activity, also described below. These
proteins, biologically active homologues thereof, and nucleic acid
molecules, as well as other acyltransferase proteins, homologues
thereof, and nucleic acid molecules, are encompassed by the present
invention and specific examples will be discussed in detail
below.
[0254] Another embodiment of the present invention relates to an
isolated protein that utilizes PUFA-CoA as a substrate in forming
PL or TAG (e.g., 3-glycerol-phosphate acyltransferases (GPAT),
lysophosphatidic acid acyltransferases (LPAAT) and diacylglycerol
acyltransferases (DAGAT)). Preferred proteins include any of the
acyltransferases selected from GPATs, LPAATs and DAGATs. In one
aspect, the isolated proteins are derived from an organism that
endogenously expresses a PUFA PKS system (PKS synthase) or at least
a biosynthesis pathway for the production of PUFAs. Such organisms
include, but are not limited to, a Thraustochytrid or
Crypthecodinium cohnii. In one aspect, the isolated acyltransferase
is derived from Schizochytrium, Thraustochytrium, or Ulkenia. In
another aspect, the isolated acyltransferase is derived from
Schizochytrium ATCC 20888 or from Schizochytrium sp. strain N230D.
In another aspect, the acyltransferase is derived from
Crypthecodinium cohnii. In another aspect, any acyltransferase that
functions in conjunction with any PUFA PKS system to increase the
production and/or accumulation of PUFAs in a host cell or organism
can be used in the present invention. The invention is not limited
to those specific examples described herein.
[0255] In another aspect, the isolated acyl transferase is encoded
by a nucleotide sequence selected from any one of SEQ ID NOs:100,
102, 103, 105, 106, 108, 109, 111, 112, or 114-121. In another
aspect, the isolated acyltransferase is encoded by a degenerate
nucleic acid sequence encoding a protein that is encoded by a
nucleotide sequence selected from any one of SEQ ID NOs: 100, 102,
103, 105, 106, 108, 109, 111, 112, or 114-121. In yet another
aspect, the isolated acyltransferase comprises an amino acid
sequence selected from any one of SEQ ID NOs: 101, 104, 107, 110,
or 113, or a homologue of any of such amino acid sequences
(described below), including any biologically active fragments or
domains of such sequences. In a preferred embodiment, the isolated
acyltransferase comprises an amino acid sequence represented herein
by SEQ ID NO: 101, 104, 107, 110, or 113, or a homologue of such
amino acid sequence. In a more preferred embodiment, the isolated
acyltransferase comprises an amino acid sequence represented herein
by SEQ ID NO:101 or 104, or a homologue of such sequence, with SEQ
ID NO:101 being particularly preferred. Combinations of
acyltransferases described herein are also encompassed for use in
the present invention.
[0256] In yet another aspect, the isolated acyltransferase
comprises an amino acid sequence selected from any one of SEQ ID
NOs:, or a homologue of any of such amino acid sequences (described
below), including any biologically active fragments or domains of
such sequences.
[0257] The invention includes the expression of one or more
acyl-CoA synthetases as described and exemplified herein with a
PUFA synthase as described herein and with an exogenous PPTase
(alone or in combination with inhibition of endogenous PPTases),
which are utilized alone or in combination with any one or more
strategies described herein (e.g., any one, two, three, or four of:
codon optimization, organelle-targeting, enhancement of PUFA
synthase competition for malonyl CoA (e.g., by inhibition of FAS),
and/or expression of an acyl CoA synthetase), to increase PUFA
production and/or accumulation in a heterologous host.
Organelle-Specific Expression
[0258] Relating to another strategy described above, one embodiment
of the invention relates to the targeting of expression of the PUFA
synthase enzymes, the PPTase, and/or any one or more of the
accessory proteins and/or targeted genetic modifications to one or
more organelles of the host. For example, in one embodiment,
expression of the PUFA synthase system and the PPTase is targeted
to the plastid of a plant. In another embodiment, expression of the
PUFA synthase system and the PPTase is targeted to the cytosol. In
another embodiment, expression of the PUFA synthase system and the
PPTase is targeted to both the plastid and the cytosol of a plant.
In any of these embodiments, other targets can be directed to the
plastid or the cytosol. In one aspect, expression of an acyl-CoA
synthetase is targeted to the cytosol, and in another embodiment,
such expression is targeted to the plastid. In one embodiment, one
acyl-CoA synthetase is targeted to the cytosol and another acyl-CoA
synthetase is targeted to the plastid. Preferably, acyl-CoA
synthetases are expressed in the cytosol to convert the DHA and/or
DPA free fatty acids to Acyl-CoAs, which in turn can be utilized by
the acyltransferases. Acyltransferases are generally
co-translationally targeted to the endoplasmic reticulum.
Inhibition of FAS systems, such as by genetic modification to
inhibit one or more host enzymes, can be directed to the same
organelle(s) in which the PUFA synthase is expressed.
[0259] One exemplary plastid targeting sequence is derived from a
Brassica napus acyl-ACP thioesterase, the amino acid sequence of
the encoded targeting peptide being represented herein by SEQ ID
NO:81. A variety of other plastid targeting sequences are known in
the art and can be used in embodiments where the heterologous host
is a plant or plant cell, and wherein targeting to the plastid is
desired.
[0260] The invention includes the use of organelle targeting (e.g.,
to the plastid or chloroplast in plants) with expression of a PUFA
synthase as described herein and with an exogenous PPTase (alone or
in combination with inhibition of endogenous PPTases), which are
utilized alone or in combination with any one or more strategies
described herein (e.g., any one, two, three, or four of: codon
optimization, enhancement of PUFA synthase competition for malonyl
CoA (e.g., by inhibition of FAS), expression of one or more
acyl-CoA synthetases, and/or expression of one or more
acyltransferases or related enzymes), to increase PUFA production
and/or accumulation in a heterologous host.
[0261] The targeting of gene products to the plastid or chloroplast
is controlled by a signal sequence found at the amino terminal end
of various proteins and which is cleaved during import yielding the
mature protein (e.g. with regard to chloroplast targeting, see,
e.g., Comai et al., J. Biol. Chem. 263: 15104-15109 (1988)). These
signal sequences can be fused to heterologous gene products to
effect the import of heterologous products into the chloroplast
(van den Broeck et al. Nature 313: 358-363 (1985)). DNA encoding
for appropriate signal sequences can be isolated from the cDNAs
encoding the RUBISCO protein, the CAB protein, the EPSP synthase
enzyme, the GS2 protein and many other proteins which are known to
be chloroplast localized.
[0262] In various embodiments of the invention, it may be
particularly advantageous to direct the localization of proteins
employed in the invention to a subcellular compartment, for
example, to the plastid or chloroplast. Proteins can be directed to
the chloroplast by including at their amino-terminus a chloroplast
transit peptide (CTP). Similarly, proteins can be directed to the
plastid by including at their N-terminus a plastid transit or
signaling peptide.
[0263] Naturally occurring chloroplast targeted proteins,
synthesized as larger precursor proteins containing an
amino-terminal chloroplast targeting peptide directing the
precursor to the chloroplast import machinery, are well known in
the art. Chloroplast targeting peptides are generally cleaved by
specific endoproteases located within the chloroplast organelle,
thus releasing the targeted mature and preferably active enzyme
from the precursor into the chloroplast milieu. Examples of
sequences encoding peptides which are suitable for directing the
targeting of the gene or gene product to the chloroplast or plastid
of the plant cell include the petunia EPSPS CTP, the Arabidopsis
EPSPS CTP2 and intron, and others known to those skilled in the
art. Such targeting sequences provide for the desired expressed
protein to be transferred to the cell structure in which it most
effectively functions, or by transferring the desired expressed
protein to areas of the cell in which cellular processes necessary
for desired phenotypic function are concentrated. Specific examples
of chloroplast targeting peptides are well known in the art and
include the Arabidopsis thaliana ribulose bisphosphate carboxylase
small subunit ats1A transit peptide, an Arabidopsis thaliana EPSPS
transit peptide, and a Zea maize ribulose bisphosphate carboxylase
small subunit transit peptide.
[0264] An optimized transit peptide is described, for example, by
Van den Broeck et al., "Targeting of a foreign protein to
chloroplasts by fusion to the transit peptide from the small
subunit of ribulose 1,5-biphosphate carboxylase", Nature,
313:358-363 (1985). Prokaryotic and eukaryotic signal sequences are
disclosed, for example, by Michaelis et al. (1982) Ann. Rev.
Microbiol. 36, 425. Additional examples of transit peptides that
may be used in the invention include the chloroplast transit
peptides such as those described in Von Heijne et al., Plant Mol.
Biol. Rep. 9:104-126 (1991); Mazur et al., Plant Physiol. 85: 1110
(1987); Vorst et al., Gene 65: 59 (1988). Chen & Jagendorf (J.
Biol. Chem. 268: 2363-2367 (1993)) have described use of a
chloroplast transit peptide for import of a heterologous transgene.
This peptide used is the transit peptide from the rbcS gene from
Nicotiana plumbaginifolia (Poulsen et al. Mol. Gen. Genet. 205:
193-200 (1986)). One CTP that has functioned herein to localize
heterologous proteins to the chloroplast was derived from Brassica
napus acyl-ACP thioesterase.
[0265] An alternative means for localizing genes to chloroplast or
plastid includes chloroplast or plastid transformation. Recombinant
plants can be produced in which only the chloroplast DNA has been
altered to incorporate the molecules envisioned in this
application. Promoters which function in chloroplasts have been
known in the art (Hanley-Bowden et al., Trends in Biochemical
Sciences 12:67-70, 1987). Methods and compositions for obtaining
cells containing chloroplasts into which heterologous DNA has been
inserted have been described, for example by Daniell et al. (U.S.
Pat. No. 5,693,507; 1997) and Maliga et al. (U.S. Pat. No.
5,451,513; 1995).
Combinations of Strategies
[0266] According to the present invention, in the production of a
heterologous host for the production and accumulation of one or
more target PUFAs, any one or more (any combination) of the
strategies described herein for improving the production and/or
accumulation of PUFAs in the host can be used. Indeed, it is
anticipated that various combinations of strategies will be
additive or synergistic and provide improved production and/or
accumulation of PUFAs as compared to in the absence of one or more
such strategies. Indeed, the Examples provide multiple exemplary
strategies, including a variety of combinations of strategies, for
the production of PUFAs in a host organism (both those that are
heterologous hosts and organisms that naturally express a PUFA PKS
system).
[0267] A suitable genetically modified host cell or organism for
the production of PUFAs according to the present invention has the
following base attributes. The host cell or organism expresses a
PUFA PKS system, which includes the core PUFA PKS enzymes as
described herein and a PPTase that is effective to produce PUFAs
when used with the core PUFA PKS enzymes. The PUFA PKS system
and/or the PPTase may be produced endogenously by the host cell or
organism, or expressed as heterologous proteins in the host (e.g.,
by recombinant technology). The nucleic acid molecules encoding the
core PUFA PKS enzymes and/or the PPTase may be optimized for codon
usage or better expression in the host cell or organism. The host
cell or organism may additionally be modified to express one, two,
three, or more acyl-Co synthetases, including any of those
described herein or otherwise known in the art. The host cell or
organism may additionally be modified to express one, two, three,
or more acyltransferases, including any of those described herein
or otherwise known in the art. The host cell or organism may be
additional genetically modified (or otherwise selected or produced)
to enhance the ability of the PUFA PKS system to compete for the
substrate, malonyl CoA. In one aspect, this is achieved by
selection of an organism that has this characteristic naturally or
due to a natural, selected, or directed mutation or by breeding or
other technique. In another aspect, this is achieved by selectively
inhibiting one or more enzymes in the pathway(s) that compete with
PUFA PKS for malonyl CoA, such as the FAS system. In any of the
embodiments, the targeting of the PUFA PKS or accessory proteins or
modifications can be organelle-specific, such as to the plastid of
plants.
[0268] Some preferred combinations for use in connection with a
core PUFA PKS system and PPTase include, but are not limited to:
(1) expression of one, two or more acyl-CoA synthetases; (2) FAS
inhibition (e.g., by inhibition of KASII or KASIII); (3)
combination of expression of one, two or more acyl-CoA synthetases
with FAS inhibition (e.g., by inhibition of KASII or KASIII); (4)
expression of one, two or more acyl transferases; (5) combination
of expression of one, two or more acyl-CoA synthetases; FAS
inhibition (e.g., by inhibition of KASII or KASIII); and expression
of one, two or more acyl transferases.
[0269] Some exemplary combinations of modifications illustrated
herein in plants (see Example 13) include the expression of a PUFA
PKS (e.g., from Schizochytrium) and a heterologous PPTase (e.g.,
HetI from Nostoc) with: [0270] (a) Expression of an acyl-CoA
synthetase (exemplified are ACS-1 and ACS-2); [0271] (b) FAS
inhibition (exemplified are inhibition by KASII RNAi, KAS II
antisense, KASIII RNAi, and KASIII antisense); [0272] (c)
Combination of expression of an acyl-CoA synthetase with FAS
inhibition (exemplified are expression of ACS-1 with FAS inhibition
by each of KASII RNAi, KAS II antisense, KASIII RNAi, and KASIII
antisense); [0273] (d) Expression of an acyltransferase
(exemplified is LPAAT-1); [0274] (e) Combination of expression of
an acyltransferase with expression of an acyl-CoA synthetase and
with FAS inhibition (exemplified is expression of DAGAT-1 with
expression of ACS-1, each combination with inhibition of FAS by
KASII RNAi or KASIII antisense); [0275] (f) Combination of
expression of an acyltransferase with expression of two acyl-CoA
synthetases and with FAS inhibition (exemplified is expression of
DAGAT-1 with expression of ACS-1, expression of ACS-8, each
combination with inhibition of FAS by KASII RNAi or KASIII
antisense); [0276] (g) Combination of expression of two
acyltransferases with expression of an acyl-CoA synthetase and with
FAS inhibition (exemplified is expression of DAGAT-1 and LPAAT-1
with expression of ACS-1, each combination with inhibition of FAS
by KASII RNAi or KASIII antisense); and [0277] (h) Combination of
expression of two acyltransferases with expression of two acyl-CoA
synthetases and with FAS inhibition (exemplified is expression of
DAGAT-1 and LPAAT-1 with expression of ACS-1 and ACS-8, each
combination with inhibition of FAS by KASII RNAi or KASIII
antisense).
[0278] Any plant or plant cell using these combinations of
modifications, or any other modification or combination of
modifications described herein, is encompassed by the invention.
Furthermore, any host cell or organism using any modifications or
combination of modifications described herein is encompassed by the
invention, as are any products derived from such cell or organisms,
including oils comprising the target PUFAs. All of these
embodiments of the invention apply to the discussion of any of the
genetically modified organisms and methods of producing and using
such organisms as described herein.
Genetically Modified Cells, Organisms, and Methods of Producing and
Using the Same
[0279] To produce significantly high yields of one or more desired
polyunsaturated fatty acids or other bioactive molecules, an
organism, preferably a microorganism or a plant, can be genetically
modified to alter the activity and particularly, the end product,
of the PUFA PKS system in the microorganism or plant or to
introduce a PUFA PKS system into the microorganism or plant. The
present invention relates to methods to improve or enhance the
effectiveness of such genetic modification and particularly, to
improve or enhance the production and/or accumulation of the
endproduct of a PUFA PKS system, preferably PUFA(s).
[0280] Therefore, one embodiment of the present invention relates
to a genetically modified organism, wherein the organism expresses
a PUFA PKS system, and wherein the organism has been genetically
modified to express an accessory protein as described herein for
the improvement of the production and/or accumulation of PUFAs (or
other bioactive products of the PUFA PKS system) by the host,
and/or wherein the organism has been genetically modified by any
method, including natural selection and mutation, to enhance the
ability of the PUFA PKS to compete for substrate within the host
(e.g., by inhibition of FAS pathways and other competing pathways
described herein). If the PUFA PKS system is heterologous to the
host, then the organism is also preferably genetically modified to
express a PPTase as a PUFA PKS accessory protein, which is
described in detail above. In one embodiment, the organism has been
genetically modified to express an ACoAS described herein, and
preferably an ACoAS that is derived from the same genus, species or
specific organism as the organism from which the PUFA PKS system is
derived, or is capable of catalyzing the conversion of long chain
PUFA free fatty acids (FFA) produced by the PUFA PKS system to
acyl-CoA. In another embodiment, the organism has been genetically
modified to express a protein that utilizes PUFA-CoA as substrates
in forming PL or TAG. In yet another embodiment, the organism has
been genetically modified to express both the above-described ACoAS
and a protein that utilizes PUFA-CoA as substrates in forming PL or
TAG. In one embodiment, if the PUFA PKS system is endogenous to the
host, the organism can be genetically modified to express a
heterologous accessory protein as described above that improves or
enhances the production and/or accumulation of PUFAs (or another
bioactive product of the PUFA PKS system) in the host organism,
and/or the organism can be genetically modified to increase,
optimize, or enhance the expression and/or biological activity of
such an accessory protein that is endogenously expressed by the
organism (e.g., to improve the expression or activity of an
endogenous ACoAS that operates with the endogenous PUFA PKS system
in the host). In one embodiment, the organism is genetically
modified by any method, including natural selection and mutation,
directed mutation, or random mutation and screening, etc., to
enhance the ability of the PUFA PKS to compete for substrate within
the host (e.g., by inhibition of FAS pathways and other competing
pathways described herein). In one embodiment, the FAS pathway in
the organism is inhibited. In one embodiment, KASII and/or KASIII
in the organism is inhibited. These embodiments of the invention
are described in detail above. Preferred genetically modified
organisms include genetically modified microorganisms and
genetically modified plants.
[0281] The organism can endogenously express a PUFA PKS system,
although the present invention is especially useful for enhancing
the production and/or accumulation of PUFAs in organisms that are
genetically modified to express the PUFA PKS system (heterologous
hosts). The PUFA PKS system expressed by the organism can include
any PUFA PKS system, for example, PUFA PKS systems that are
entirely derived from a particular organism (e.g., a Schizochytrium
PUFA PKS system), as well as PUFA PKS systems that are produced by
"mixing and matching" nucleic acid sequences encoding proteins
and/or domains from different PUFA PKS systems (e.g., by mixing
Schizochytrium PUFA PKS proteins and/or domains with PUFA PKS
proteins and/or domains from, e.g., Thraustochytrium, Ulkenia,
Shewanella, Moritella, and/or Photobacterium, etc.) and/or from
different non-PUFA PKS systems (e.g., type I modular, type I
iterative, type II or type III PKS systems), where the proteins
and/or domains from different organisms are combined to form a
complete, functional PUFA PKS system. PUFA PKS systems, including
combining PUFA PKS genes or proteins from different organisms, are
described in detail in U.S. Pat. No. 6,140,486; U.S. Pat. No.
6,566,583; Metz et al., Science 293:290-293 (2001); U.S. Patent
Application Publication No. 20020194641; U.S. Patent Application
Publication No. 20040235127; U.S. Patent Application Publication
No. 20050100995; and PCT Publication No. WO 2006/135866; supra).
PUFA PKS genes and proteins are also disclosed in: PCT Patent
Publication No. WO 05/097982; and U.S. Patent Application
Publication No. 20050014231. Each of the above-identified
disclosures, and the genes and proteins described therein, is
incorporated herein by reference.
[0282] Accordingly, encompassed by the present invention are
methods to genetically modify organisms by: genetically modifying
at least one nucleic acid sequence in the organism that encodes at
least one functional domain or protein (or biologically active
fragment or homologue thereof) of a PUFA PKS system, including, but
not limited to, any PUFA PKS system specifically described herein,
and/or by expressing at least one recombinant nucleic acid molecule
comprising a nucleic acid sequence encoding such domain or protein.
In addition, the methods include genetically modifying the
organisms by genetically modifying at least one nucleic acid
sequence in the organism that encodes an ACoAS and/or a protein
that utilizes PUFA-CoA as substrates in forming PL or TAG at least
one functional domain or protein, and/or by expressing at least one
recombinant nucleic acid molecule comprising a nucleic acid
sequence encoding such protein(s). The methods can further include
genetically modifying the organism to inhibit a pathway that
competes with the PUFA PKS for substrate, such as the FAS system,
including, but not limited to, inhibition of KASII or KASIII in the
organism. In one embodiment, any of the exogenously introduced
nucleic acid sequences can be optimized for codon usage or improved
expression in the host. In one embodiment, any of the introduced
nucleic acid sequences can be targeted to one or more organelles in
the organism. Various embodiments of such sequences, methods to
genetically modify an organism, specific modifications, and
combinations thereof have been described in detail above and are
encompassed here. Typically, the method is used to produce a
particular genetically modified organism that produces a particular
bioactive molecule or molecules. Preferably the genetically
modified organism is a genetically modified microorganism or a
genetically modified plant.
[0283] Preferably, a genetically modified organism of the invention
produces one or more polyunsaturated fatty acids including, but not
limited to, 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), ALA (C18:3, n-3), and/or
SDA (C18:4, n-3)), and more preferably, one or more longer chain
PUFAs, including, but not limited to, EPA (C20:5, n-3), DHA (C22:6,
n-3), DPA (C22:5, n-6 or n-3), or DTA (C22:4, n-6), or any
combination thereof. In a particularly preferred embodiment, a
genetically modified plant of the invention produces one or more
polyunsaturated fatty acids including, but not limited to, EPA
(C20:5, n-3), DHA (C22:6, n-3), and/or DPA (C22:5, n-6 or n-3), or
any combination thereof.
[0284] According to the present invention, a genetically modified
organism includes an organism that has been modified using
recombinant technology or by classical mutagenesis and screening
techniques. As used herein, genetic modifications that result in a
decrease in gene expression, in the function of the gene, or in the
function of the gene product (i.e., the protein encoded by the
gene) can be referred to as inactivation (complete or partial),
deletion, interruption, blockage or down-regulation of a gene. For
example, a genetic modification in a gene which results in a
decrease in the function of the protein encoded by such gene, can
be the result of a complete deletion of the gene (i.e., the gene
does not exist, and therefore the protein does not exist), a
mutation in the gene which results in incomplete or no translation
of the protein (e.g., the protein is not expressed), or a mutation
in the gene which decreases or abolishes the natural function of
the protein (e.g., a protein is expressed which has decreased or no
enzymatic activity or action). Genetic modifications that result in
an increase in gene expression or function can be referred to as
amplification, overproduction, overexpression, activation,
enhancement, addition, or up-regulation of a gene.
[0285] The genetic modification of an organism according to the
present invention preferably affects the activity of the PUFA PKS
system expressed by the organism, whether the PUFA PKS system is
endogenous and genetically modified, endogenous with the
introduction of recombinant nucleic acid molecules into the
organism (with the option of modifying the endogenous system or
not), or provided completely by recombinant technology. To alter
the PUFA production profile of a PUFA PKS system or organism
expressing such system includes causing any detectable or
measurable change in the production of any one or more PUFAs (or
other bioactive molecule produced by the PUFA PKS system) by the
host organism as compared to in the absence of the genetic
modification (i.e., as compared to the unmodified, wild-type
organism or the organism that is unmodified at least with respect
to PUFA synthesis--i.e., the organism might have other
modifications not related to PUFA synthesis). To affect the
activity of a PUFA PKS system includes any genetic modification
that causes any detectable or measurable change or modification in
the PUFA PKS system expressed by the organism as compared to in the
absence of the genetic modification. A detectable change or
modification in the PUFA PKS system can include, but is not limited
to: a change or modification (introduction of, increase or
decrease) of the expression and/or biological activity of any one
or more of the domains in a modified PUFA PKS system as compared to
the endogenous PUFA PKS system in the absence of genetic
modification; the introduction of PUFA PKS system activity (i.e.,
the organism did not contain a PKS system or a PUFA PKS system
prior to the genetic modification) into an organism such that the
organism now has measurable/detectable PUFA PKS system
activity.
[0286] It should be noted that reference to increasing the activity
of a functional domain or protein in a PUFA PKS system, including
in an accessory protein to a PUFA PKS system, refers to any genetic
modification in the organism containing the domain or protein (or
into which the domain or protein is to be introduced) which results
in increased functionality of the domain or protein or system and
can include higher activity of the domain or protein or system
(e.g., specific activity or in vivo enzymatic activity), reduced
inhibition or degradation of the domain or protein or system, and
overexpression of the domain or protein or system. For example,
gene copy number can be increased, expression levels can be
increased by use of a promoter that gives higher levels of
expression than that of the native promoter, or a gene can be
altered by genetic engineering or classical mutagenesis to increase
the activity of the domain or protein encoded by the gene.
[0287] Similarly, reference to decreasing the activity of a
functional domain or protein in a PUFA PKS system, including in an
accessory protein to a PUFA PKS system, refers to any genetic
modification in the organism containing such domain or protein (or
into which the domain or protein is to be introduced) which results
in decreased functionality of the domain or protein and includes
decreased activity of the domain or protein, increased inhibition
or degradation of the domain or protein and a reduction or
elimination of expression of the domain or protein. For example,
the action of domain or protein of the present invention can be
decreased by blocking or reducing the production of the domain or
protein, "knocking out" the gene or portion thereof encoding the
domain or protein, reducing domain or protein activity, or
inhibiting the activity of the domain or protein. Blocking or
reducing the production of a domain or protein can include placing
the gene encoding the domain or protein under the control of a
promoter that requires the presence of an inducing compound in the
growth medium. By establishing conditions such that the inducer
becomes depleted from the medium, the expression of the gene
encoding the domain or protein (and therefore, of protein
synthesis) could be turned off. The present inventors demonstrate
the ability to delete (knock out) targeted genes in a
Thraustochytrid microorganism in the Examples section. Blocking or
reducing the activity of domain or protein could also include using
an excision technology approach similar to that described in U.S.
Pat. No. 4,743,546, incorporated herein by reference. To use this
approach, the gene encoding the protein of interest is cloned
between specific genetic sequences that allow specific, controlled
excision of the gene from the genome. Excision could be prompted
by, for example, a shift in the cultivation temperature of the
culture, as in U.S. Pat. No. 4,743,546, or by some other physical
or nutritional signal.
Genetically Modified Microorganisms
[0288] As used herein, a genetically modified microorganism can
include a genetically modified bacterium, protist, microalgae,
algae, fungus, or other microbe. Such a genetically modified
microorganism has a genome which is modified (i.e., mutated or
changed) from its normal (i.e., wild-type or naturally occurring)
form such that the desired result is achieved (i.e., increased or
modified PUFA PKS activity and/or production and accumulation of a
desired product using the PUFA PKS system). Genetic modification of
a microorganism can be accomplished using classical strain
development and/or molecular genetic techniques. Such techniques
known in the art and are generally disclosed for microorganisms,
for example, in Sambrook et al., 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Labs Press. The reference
Sambrook et al., ibid., is incorporated by reference herein in its
entirety. A genetically modified microorganism can include a
microorganism in which nucleic acid molecules have been inserted,
deleted or modified (i.e., mutated; e.g., by insertion, deletion,
substitution, and/or inversion of nucleotides), in such a manner
that such modifications provide the desired effect within the
microorganism.
[0289] Examples of suitable host microorganisms for genetic
modification include, but are not limited to, yeast including
Saccharomyces cerevisiae, Saccharomyces carlsbergensis, or other
yeast such as Candida, Kluyveromyces, or other fungi, for example,
filamentous fungi such as Aspergillus, Neurospora, Penicillium,
etc. Bacterial cells also may be used as hosts. These include, but
are not limited to, Escherichia coli, which can be useful in
fermentation processes. Alternatively, and only by way of example,
a host such as a Lactobacillus species or Bacillus species can be
used as a host.
[0290] Other hosts for use in the present invention include
microorganisms from a genus including, but not limited to:
Thraustochytrium, Japonochytrium, Aplanochytrium, Elina and
Schizochytrium within the Thraustochytriaceae, and Labyrinthula,
Labyrinthuloides, and Labyrinthomyxa within the Labyrinthulaceae.
Preferred species within these genera include, but are not limited
to: any species described below. Particularly preferred strains of
Thraustochytriales include, but are not limited to: Schizochytrium
sp. (S31)(ATCC 20888); Schizochytrium sp. (S8)(ATCC 20889);
Schizochytrium sp. (LC-RM)(ATCC 18915); Schizochytrium sp. (SR21);
Schizochytrium sp. N230D, Schizochytrium aggregatum (Goldstein et
Belsky)(ATCC 28209); Schizochytrium limacinum (Honda et
Yokochi)(IFO 32693); Thraustochytrium sp. (23B)(ATCC 20891);
Thraustochytrium striatum (Schneider)(ATCC 24473); Thraustochytrium
aureum (Goldstein)(ATCC 34304); Thraustochytrium roseum
(Goldstein)(ATCC 28210); and Japonochytrium sp. (L1)(ATCC
28207).
[0291] According to the present invention, the term
"thraustochytrid" refers to any members of the order
Thraustochytriales, which includes the family Thraustochytriaceae,
and the term "labyrinthulid" refers to any member of the order
Labyrinthulales, which includes the family Labyrinthulaceae. The
members of the family Labyrinthulaceae were at one time considered
to be members of the order Thraustochytriales, but in more recent
revisions of the taxonomy of such organisms, the family is now
considered to be a member of the order Labyrinthulales, and both
Labyrinthulales and Thraustochytriales are considered to be members
of the phylum Labyrinthulomycota. Developments have resulted in
frequent revision of the taxonomy of the thraustochytrids and
labyrinthulids. However, taxonomic theorists now generally place
both of these groups of microorganisms with the algae or algae-like
protists within the Stramenopile lineage. The current taxonomic
placement of the thraustochytrids and labyrinthulids can be
summarized as follows: [0292] Realm: Stramenopila (Chromista)
[0293] Phylum: Labyrinthulomycota [0294] Class: Labyrinthulomycetes
[0295] Order: Labyrinthulales [0296] Family: Labyrinthulaceae
[0297] Order: Thraustochytriales [0298] Family:
Thraustochytriaceae
[0299] However, because of remaining taxonomic uncertainties it
would be best for the purposes of the present invention to consider
the strains described in the present invention as thraustochytrids
to include the following organisms: Order: Thraustochytriales;
Family: Thraustochytriaceae; Genera: Thraustochytrium (Species:
sp., arudimentale, aureum, benthicola, globosum, kinnei, motivum,
multirudimentale, pachydermum, proliferum, roseum, striatum),
Ulkenia (Species: sp., amoeboidea, kerguelensis, minuta, profunda,
radiata, sailens, sarkariana, schizochytrops, visurgensis,
yorkensis), Schizochytrium (Species: sp., aggregatum, limnaceum,
mangrovei, minutum, octosporum), Japonochytrium (Species: sp.,
marinum), Aplanochytrium (Species: sp., haliotidis, kerguelensis,
profunda, stocchinoi), Althornia (Species: sp., crouchii), or Elina
(Species: sp., marisalba, sinorifica). It is to be noted that the
original description of the genus Ulkenia was not published in a
peer-reviewed journal so some questions remain as to the validity
of this genus and the species placed within it. For the purposes of
this invention, species described within Ulkenia will be considered
to be members of the genus Thraustochytrium.
[0300] Strains described in the present invention as Labyrinthulids
include the following organisms: Order: Labyrinthulales, Family:
Labyrinthulaceae, Genera: Labyrinthula (Species: sp., algeriensis,
coenocystis, chattonii, macrocystis, macrocystis atlantica,
macrocystis macrocystis, marina, minuta, roscoffensis, valkanovii,
vitellina, vitellina pacifica, vitellina vitellina, zopfii),
Labyrinthuloides (Species: sp., haliotidis, yorkensis),
Labyrinthomyxa (Species: sp., marina), Diplophrys (Species: sp.,
archeri), Pyrrhosorus (Species: sp., marinus), Sorodiplophrys
(Species: sp., stercorea) or Chlamydomyxa (Species: sp.,
labyrinthuloides, montana) (although there is currently not a
consensus on the exact taxonomic placement of Pyrrhosorus,
Sorodiplophrys or Chlamydomyxa).
[0301] In one embodiment of the present invention, the endogenous
PUFA PKS system and/or the endogenous PUFA PKS accessory proteins
(e.g., ACoAS) of a microorganism is genetically modified by, for
example, classical mutagenesis and selection techniques and/or
molecular genetic techniques, include genetic engineering
techniques. Genetic engineering techniques can include, for
example, using a targeting recombinant vector to delete a portion
of an endogenous gene or to replace a portion of an endogenous gene
with a heterologous sequence. Examples of heterologous sequences
that could be introduced into a host genome include sequences
encoding at least one functional PUFA PKS domain or protein from
another PKS system or even an entire PUFA PKS system (e.g., all
genes associated with the PUFA PKS system). A heterologous sequence
can also include a sequence encoding a modified functional domain
(a homologue) of a natural domain from a PUFA PKS system. Other
heterologous sequences that can be introduced into the host genome
include nucleic acid molecules encoding proteins that affect the
activity of the endogenous PUFA PKS system, such as the accessory
proteins described herein. For example, one could introduce into
the host genome a nucleic acid molecule encoding a ACoAS, and
particularly, an ACoAS that enhances the production and/or
accumulation of PUFAs in the host as compared to the endogenous
ACoAS that operates with the PUFA PKS system.
Genetically Modified Plants
[0302] Another embodiment of the present invention relates to a
genetically modified plant, wherein the plant has been genetically
modified to recombinantly express a PUFA PKS system, including a
PPTase, as described herein, and wherein the plant has been further
genetically modified to express an accessory protein as described
herein for the improvement of the production and/or accumulation of
PUFAs (or other bioactive products of the PUFA PKS system) by the
host and/or to inhibit pathways that compete with the PUFA PKS
system (e.g., inhibition of the FAS system). Preferably, such
accessory protein is an ACoAS and/or a protein that utilizes
PUFA-CoA as substrates in forming PL or TAG (e.g., a GPAT, LFAAT,
or DAGAT).
[0303] As used herein, a genetically modified plant can include any
genetically modified plant including higher plants and
particularly, any consumable plants or plants useful for producing
a desired bioactive molecule (e.g., PUFA) of the present invention.
"Plant parts", as used herein, include any parts of a plant,
including, but not limited to, seeds (including mature seeds and
immature seeds), pollen, embryos, flowers, fruits, shoots, leaves,
roots, stems, explants, etc. A genetically modified plant has a
genome which is modified (i.e., mutated or changed) from its normal
(i.e., wild-type or naturally occurring) form such that the desired
result is achieved (i.e., increased or modified PUFA PKS activity
and/or production and/or accumulation of a desired product using
the PUFA PKS system). Genetic modification of a plant can be
accomplished using classical strain development and/or molecular
genetic techniques. Methods for producing a transgenic plant,
wherein a recombinant nucleic acid molecule encoding a desired
amino acid sequence is incorporated into the genome of the plant,
are known in the art. A preferred plant to genetically modify
according to the present invention is preferably a plant suitable
for consumption by animals, including humans.
[0304] Preferred plants to genetically modify according to the
present invention (i.e., plant host cells) include, but are not
limited to any higher plants, including both dicotyledonous and
monocotyledonous plants, and particularly consumable plants,
including crop plants and especially plants used for their oils.
Such plants can include, but are not limited to, for example:
canola, soybeans, rapeseed, linseed, corn, safflowers, sunflowers
and tobacco. Thus, any plant species or plant cell may be selected.
Particular cells used herein, and plants grown or derived
therefrom, include, but are not limited to, cells obtainable from
canola (Brassica rapa L.); soybean (Glycine max); rapeseed
(Brassica spp.); linseed/flax (Linum usitatissimum); maize (corn)
(Zea mays); safflower (Carthamus tinctorius); sunflower (Helianthus
annuus); tobacco (Nicotiana tabacum); Arabidopsis thaliana, Brazil
nut (Betholettia excelsa); castor bean (Riccinus communis); coconut
(Cocus nucifera); coriander (Coriandrum sativum); cotton (Gossypium
spp.); groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis);
mustard (Brassica spp. and Sinapis alba); oil palm (Elaeis
guineeis); olive (Olea eurpaea); rice (Oryza sativa); squash
(Cucurbita maxima); barley (Hordeum vulgare); wheat (Traeticum
aestivum); and duckweed (Lemnaceae sp.). It should be noted that in
accordance herewith the genetic background within a plant species
may vary.
[0305] Plant lines from these plants, optimized for a particularly
desirable trait, e.g. disease resistance, ease of plant
transformation, oil content or profile, etc., may be produced,
selected or identified in accordance herewith. Preferred plant
lines may be selected through plant breeding, or through methods
such as marker assisted breeding and tilling. It should be noted
that plant lines displaying modulated activity with respect to any
of the herein mentioned accessory proteins, targeted inhibition of
pathways, and/or the PUFA PKS system (PUFA synthase) are
particularly useful.
[0306] In a further embodiment plant cell cultures may be used in
accordance herewith. In such embodiments plant cells are not grown
into differentiated plants and cultivated using ordinary
agricultural practices, but instead grown and maintained in a
liquid medium.
[0307] Other preferred plants include those plants that are known
to produce compounds used as pharmaceutical agents, flavoring
agents, nutraceutical agents, functional food ingredients or
cosmetically active agents or plants that are genetically
engineered to produce these compounds/agents.
[0308] As discussed above, the PUFA PKS synthase of the present
invention does not utilize the fatty acid products of FAS systems.
Instead, it produces the final PUFA product (the primary PUFA
product) from the same small precursor molecule that is utilized by
FASs and elongases (malonyl-CoA). Therefore, intermediates in the
synthesis cycle are not released in any significant amount, and the
PUFA product (also referred to herein as the primary PUFA product)
is efficiently transferred to phospholipids (PL) and
triacylglycerol (TAG) fractions of the lipids. Indeed, a PUFA PKS
system may produce two target or primary PUFA products (e.g., the
PUFA PKS system from Schizochytrium produces both DHA and DPA n-6
as primary products), but DPA is not an intermediate in the pathway
to produce DHA. Rather, each is a separate product of the same PUFA
PKS system. Therefore, PUFA PKS genes are an excellent means of
producing oils containing PUFAs, and particularly, long chain PUFAs
(LCPUFAs) in a heterologous host, such as a plant, wherein the oils
are substantially free (defined below) of the intermediates and
side products that contaminate oils produced by the "standard" PUFA
pathway (also defined below).
[0309] Therefore, it is an object of the present invention to
produce, via the genetic manipulation of plants as described
herein, polyunsaturated fatty acids of desired chain length and
with desired numbers of double bonds and, by extension, oil seed
and oils obtained from such plants (i.e., obtained from the oil
seeds of such plants) comprising these PUFAs. Examples of PUFAs
that can be produced by the present invention include, but are not
limited to, DHA (docosahexaenoic acid (C22:6, n-3)), ARA
(eicosatetraenoic acid or arachidonic acid (C20:4, n-6)), DPA
(docosapentaenoic acid (C22:5, n-6 or n-3)), and EPA
(eicosapentaenoic acid (C20:5, n-3)), and any combinations thereof.
The present invention allows for the production of commercially
valuable lipids enriched in one or more desired (target or primary)
PUFAs by the present inventors' development of genetically modified
plants through the use of the polyketide synthase-like system that
produces PUFAs.
[0310] According to the present invention, reference to a "primary
PUFA", "target PUFA", "intended PUFA", or "desired PUFA" refers to
the particular PUFA or PUFAs that are the intended or targeted
product of the enzyme pathway that is used to produce the PUFA(s).
For example, when using elongases and desaturases to modify
products of the FAS system, one can select particular combinations
of elongases and desaturases that, when used together, will produce
a target or desired PUFA (e.g., DHA or EPA). As discussed above,
such target or desired PUFA produced by the standard pathway may
not actually be a "primary" PUFA in terms of the amount of PUFA as
a percentage of total fatty acids produced by the system, due to
the formation of intermediates and side products that can actually
represent the majority of products produced by the system. However,
one may use the term "primary PUFA" even in that instance to refer
to the target or intended PUFA product produced by the elongases or
desaturases used in the system.
[0311] When using a PUFA PKS system as preferred in the present
invention, a given PUFA PKS system derived from a particular
organism will produce particular PUFA(s), such that selection of a
PUFA PKS system from a particular organism will result in the
production of specified target or primary PUFAs. For example, use
of a PUFA PKS system from Schizochytrium will result in the
production of DHA and DPAn-6 as the target or primary PUFAs. Use of
a PUFA PKS system from various Shewanella species, on the other
hand, will result in the production of EPA as the target or primary
PUFA. It is noted that the ratio of the primary or target PUFAs can
differ depending on the selection of the particular PUFA PKS system
and on how that system responds to the specific conditions in which
it is expressed. For example, use of a PUFA PKS system from
Thraustochytrium 23B (ATCC No. 20892) will also result in the
production of DHA and DPAn-6 as the target or primary PUFAs;
however, in the case of Thraustochytrium 23B, the ratio of DHA to
DPAn-6 is about 10:1 (and can range from about 8:1 to about 40:1),
whereas in Schizochytrium, the ratio is typically about 2.5:1.
Therefore, use of a Thraustochytrium PUFA PKS system or proteins or
domains can alter the ratio of PUFAs produced by an organism as
compared to Schizochytrium even though the target PUFAs are the
same. In addition, as discussed below, one can also modify a given
PUFA PKS system by intermixing proteins and domains from different
PUFA PKS systems or PUFA PKS and PKS systems, or one can modify a
domain or protein of a given PUFA PKS system to change the target
PUFA product and/or ratios.
[0312] According to the present invention, reference to
"intermediate products" or "side products" of an enzyme system that
produces PUFAs refers to any products, and particularly, fatty acid
products, that are produced by the enzyme system as a result of the
production of the target or primary PUFA(s) of the system, but
which are not the primary or target PUFA(s). In one embodiment,
intermediate and side products may include non-target fatty acids
that are naturally produced by the wild-type plant, or by the
parent plant used as a recipient for the indicated genetic
modification, but are now classified as intermediate or side
products because they are produced in greater levels as a result of
the genetic modification, as compared to the levels produced by the
wild-type plant, or by the parent plant used as a recipient for the
indicated genetic modification. Intermediate and side products are
particularly significant in the standard pathway for PUFA synthesis
and are substantially less significant in the PUFA PKS pathway, as
discussed above. It is noted that a primary or target PUFA of one
enzyme system may be an intermediate of a different enzyme system
where the primary or target product is a different PUFA, and this
is particularly true of products of the standard pathway of PUFA
production, since the PUFA PKS system substantially avoids the
production of intermediates. For example, when using the standard
pathway to produce EPA, fatty acids such as GLA, DGLA and SDA are
produced as intermediate products in significant quantities (e.g.,
U.S. Patent Application Publication 2004/0172682 illustrates this
point). Similarly, and also illustrated by U.S. Patent Application
Publication 2004/0172682, when using the standard pathway to
produce DHA, in addition to the fatty acids mentioned above, ETA
and EPA (notably the target PUFA in the first example above) are
produced in significant quantities and in fact, may be present in
significantly greater quantities relative to the total fatty acid
product than the target PUFA itself. This latter point is also
shown in U.S. Patent Application Publication 2004/0172682, where a
plant that was engineered to produce DHA by the standard pathway
produces more EPA as a percentage of total fatty acids than the
targeted DHA.
[0313] To produce significantly high yields of one or more desired
polyunsaturated fatty acids, a plant can be genetically modified to
introduce a PUFA PKS system into the plant. Plants are not known to
endogenously contain a PUFA PKS system, and therefore, the PUFA PKS
systems of the present invention represent an opportunity to
produce plants with unique fatty acid production capabilities. It
is a particularly preferred embodiment of the present invention to
genetically engineer plants to produce one or more PUFAs in the
same plant, including, EPA, DHA, DPA (n3 or n6), ARA, GLA, SDA and
others, including any combination thereof. The present invention
offers the ability to create any one of a number of "designer oils"
in various ratios and forms. Moreover, the disclosure of the PUFA
PKS genes from the particular marine organisms described herein
offer the opportunity to more readily extend the range of PUFA
production and successfully produce such PUFAs within temperature
ranges used to grow most crop plants.
[0314] Therefore, one embodiment of the present invention relates
to a genetically modified plant or part of a plant (e.g., wherein
the plant has been genetically modified to express a PUFA PKS
system described herein), which includes the core PUFA PKS enzyme
complex and a PPTase, as described herein, wherein the plant has
been further genetically modified to express an accessory protein
as described herein for the improvement of the production and/or
accumulation of PUFAs (or other bioactive products of the PUFA PKS
system) by the host and/or wherein the plant has been genetically
modified to inhibit pathways that compete with the PUFA PKS system
(e.g., inhibition of the FAS system) as described herein.
Preferably, such accessory protein is an ACoAS and/or a protein
that utilizes PUFA-CoA as substrates in forming PL or TAG (e.g., a
GPAT, LFAAT, or DAGAT). so that the plant produces PUFAs.
[0315] Preferably, such additional genetic modification is any
modification (naturally occurring, selected, or synthesized) that
increases the flux through the PUFA synthase pathway by reducing
competition for the malonyl-CoA pool(s). There are many possible
ways to achieve enhanced ability to compete for this substrate.
These include, but are not limited to, 1) inhibition of competing
pathways, including inhibition of any elements in the FAS pathway,
e.g., by reducing expression levels of enzymes or subunits involved
in those pathways (e.g., by use of antisense RNA, RNAi,
co-suppression, or mutations), 2) expression of the PUFA synthase
in heterologous hosts in which competing pathways have been reduced
or blocked (e.g., in Canola where the ability to elongate fatty
acids in the cytoplasm has been blocked), and/or 3) by increasing
the pool of malonyl-CoA (e.g., by expression of acetyl-CoA
carboxylase). In one embodiment, KASII and/or KASIII are inhibited
in the plant (e.g., by RNAi or by antisense).
[0316] As discussed above, the genetically modified plant useful in
the present invention has been genetically modified to express a
PUFA PKS system. The PUFA PKS system can include any PUFA PKS
system, such as any PUFA PKS system described in, for example, U.S.
Pat. No. 6,566,583; U.S. Patent Application Publication No.
20020194641; U.S. Patent Application Publication No. 20040235127;
U.S. Patent Application Publication No. 20050100995; and PCT
Publication No. WO 2006/135866. The PUFA PKS system can be chosen
from, but is not limited to, any of the specific PUFA PKS systems
identified and characterized in these patents and patent
publications, such as the PUFA PKS systems from Schizochytrium sp.
American Type Culture Collection (ATCC) No. 20888, and mutant
strains derived therefrom (e.g., strain N230D); Thraustochytrium
23B ATCC No. 20892, and mutant strains derived therefrom;
Shewanella olleyana Australian Collection of Antarctic
Microorganisms (ACAM) strain number 644, and mutant strains derived
therefrom; or Shewanella japonica ATCC strain number BAA-316, and
mutant strains derived therefrom.
[0317] In one embodiment, the PUFA PKS system comprises domains
selected from any of the above PUFA PKS systems, wherein the
domains are combined (mixed and matched) to form a complete PUFA
PKS system meeting the minimum requirements as discussed above. The
plant can also be further modified with at least one domain or
biologically active fragment thereof of another PKS system,
including, but not limited to, Type I PKS systems (iterative or
modular), Type II PKS systems, and/or Type III PKS systems, which
may substitute for a domain in a PUFA PKS system. Finally, any of
the domains of a PUFA PKS system can be modified from their natural
structure to modify or enhance the function of that domain in the
PUFA PKS system (e.g., to modify the PUFA types or ratios thereof
produced by the system). Such mixing of domains to produce chimeric
PUFA PKS proteins is described in the patents and patent
publications referenced above.
[0318] Preferably, a plant having any of the above-identified
characteristics is a plant that has been genetically modified to
express a PUFA PKS system (PUFA synthase) as described in detail
herein (i.e., the PUFA PKS system is the enzyme system that
produces the target PUFA(s) in the plant). In one embodiment, the
plant has been genetically modified to express a PUFA PKS system
comprised of PUFA PKS proteins/domains from a thraustochytrid,
including, but not limited to, Schizochytrium, Thraustochytrium,
Ulkenia, Japonochytrium, Aplanochytrium, Althornia, or Elina. In
one embodiment, the plant has been genetically modified to express
a PUFA PKS system comprised of PUFA PKS proteins/domains from a
labrynthulid. In another embodiment, the plant has been genetically
modified to express a PUFA PKS system comprised of PUFA PKS
proteins/domains from a marine bacterium, including, but not
limited to, Shewanella japonica or Shewanella olleyana. In one
embodiment, the plant has been genetically modified to express a
PUFA PKS system comprised of Schizochytrium OrfsA, B and C
(including homologues or synthetic versions thereof), and a PPTase
(e.g., HetI) as described above (e.g., see SEQ ID NOs:1-32 and SEQ
ID NO:33, and discussion of Schizochytrium PUFA PKS system above).
In another embodiment, the plant has been genetically modified to
express a PUFA PKS system comprised of Thraustochytrium OrfsA, B
and C (including homologues or synthetic versions thereof), and a
PPTase (e.g., HetI) as described above (e.g., see SEQ ID NOs:38-68
and SEQ ID NO:33, and discussion of Thraustochytrium PUFA PKS
system above; see also U.S. Patent Application Publication No.
20050014231). In another embodiment, the plant has been genetically
modified to express a PUFA PKS system comprised of other
thraustochytrid OrfsA, B and C (including homologues or synthetic
versions thereof), and a PPTase (e.g., HetI) (e.g., see PCT Patent
Publication No. WO 05/097982). In another embodiment, the plant has
been genetically modified to express a PUFA PKS system comprised of
PUFA PKS Orfs from marine bacteria such as Shewanella (including
homologues or synthetic versions thereof), and a PPTase (e.g., the
endogenous Shewanella PPTase) as described above (e.g., see SEQ ID
NOs:1-6 for Shewanella japonica, SEQ ID NOs: 7-12 for Shewanella
olleyana). In another embodiment, the plant has been genetically
modified to express any combinations of domains and proteins from
such PUFA PKS systems (e.g., a chimeric PUFA PKS system).
[0319] Finally, as discussed above, the genetic modification of the
plant may include the introduction of one or more accessory
proteins that will work with the core PUFA PKS enzyme complex to
enable, facilitate, or enhance production of PUFAs by the plant,
and/or a genetic modification that results in enhanced flux of
malonyl CoA substrate through the PUFA PKS system, such as by any
inhibition of the FAS system described herein, or the use of other
strategies for achieving the same result as described herein. The
genetic modification of the plant can also include the optimization
of genes for preferred host codon usage, as well as targeting of
the PUFA synthase enzymes to particular organelles (e.g., the
plastid).
[0320] Preferably, the plant is an oil seed plant, wherein the oil
seeds, and/or the oil in the oil seeds, contain PUFAs produced by
the PUFA PKS system. Such oils contain a detectable amount of at
least one target or primary PUFA that is the product of the PUFA
PKS system. Additionally, such oils are substantially free of
intermediate or side products that are not the target or primary
PUFA products and that are not naturally produced by the endogenous
FAS system in the wild-type plants (i.e., wild-type plants produce
some shorter or medium chain PUFAs, such as 18 carbon PUFAs, via
the FAS system, but there will be new, or additional, fatty acids
produced in the plant as a result of genetic modification with a
PUFA PKS system). In other words, as compared to the profile of
total fatty acids from the wild-type plant (not genetically
modified) or the parent plant used as a recipient for the indicated
genetic modification, the majority of additional fatty acids (new
fatty acids or increased fatty acids resulting from the genetic
modification) in the profile of total fatty acids produced by
plants that have been genetically modified with a PUFA PKS system,
comprise the target or intended PUFA products of the PUFA PKS
system (i.e., the majority of additional, or new, fatty acids in
the total fatty acids that are produced by the genetically modified
plant are the target PUFA(s)).
[0321] Furthermore, to be "substantially free" of intermediate or
side products of the system for synthesizing PUFAs, or to not have
intermediate or side products present in substantial amounts, means
that any intermediate or side product fatty acids (non-target
PUFAs) that are produced in the genetically modified plant (and/or
parts of plants and/or seed oil fraction) as a result of the
introduction or presence of the enzyme system for producing PUFAs
(i.e., that are not produced by the wild-type plant or the parent
plant used as a recipient for the indicated genetic modification),
are present in a quantity that is less than about 10% by weight of
the total fatty acids produced by the plant, and more preferably
less than about 9%, and more preferably less than about 8%, and
more preferably less than about 7%, and more preferably less than
about 6%, and more preferably less than about 5%, and more
preferably less than about 4%, and more preferably less than about
3%, and more preferably less than about 2%, and more preferably
less than about 1% by weight of the total fatty acids produced by
the plant, and more preferably less than about 0.5% by weight of
the total fatty acids produced by the plant.
[0322] In a preferred embodiment, to be "substantially free" of
intermediate or side products of the system for synthesizing PUFAs,
or to not have intermediate or side products present in substantial
amounts, means that any intermediate or side product fatty acids
that are produced in the genetically modified plant (and/or parts
of plants and/or in seed oil fraction) as a result of the enzyme
system for producing PUFAS (i.e., that are not produced by the
wild-type plant or by the parent plant used as a recipient for the
indicated genetic modification for production of target PUFAs), are
present in a quantity that is less than about 10% by weight of the
total additional fatty acids produced by the plant (additional
fatty acids being defined as those fatty acids or levels of fatty
acids that are not naturally produced by the wild-type plant or by
the parent plant that is used as a recipient for the indicated
genetic modification for production of target PUFAs), and more
preferably less than about 9%, and more preferably less than about
8%, and more preferably less than about 7%, and more preferably
less than about 6%, and more preferably less than about 5%, and
more preferably less than about 4%, and more preferably less than
about 3%, and more preferably less than about 2%, and more
preferably less than about 1% of the total additional fatty acids
produced by the plant. Therefore, in contrast to the fatty acid
profile of plants that have been genetically modified to produce
PUFAs via the standard pathway, the majority of fatty acid products
resulting from the genetic modification with a PUFA PKS system will
be the target or intended fatty acid products.
[0323] When the target product of a PUFA PKS system is a long chain
PUFA, such as DHA, DPA (n-6 or n-3), or EPA, intermediate products
and side products that are not present in substantial amounts in
the total lipids of plants genetically modified with such PUFA PKS
can include, but are not limited to: gamma-linolenic acid (GLA;
18:3, n-6); stearidonic acid (STA or SDA; 18:4, n-3);
dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6), arachidonic
acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA; 20:3, n-9) and
various other intermediate or side products, such as 20:0; 20:1
(.DELTA.5); 20:1 (.DELTA.11); 20:2 (.DELTA.8,11); 20:2
(.DELTA.11,14); 20:3 (.DELTA.5,11,14); 20:3 (.DELTA.11,14,17); mead
acid (20:3; .DELTA.5,8,11); or 20:4 (.DELTA.5,1,14,17). In
addition, when the target product is a particular PUFA, such as
DHA, the intermediate products and side products that are not
present in substantial amounts in the total lipids of the
genetically modified plants also include other PUFAs, including
other PUFAs that are a natural product of a different PUFA PKS
system, such as EPA in this example. It is to be noted that the
PUFA PKS system of the present invention can also be used, if
desired, to produce as a target PUFA a PUFA that can include GLA,
SDA or DGLA.
[0324] Using the knowledge of the genetic basis and domain
structure of PUFA PKS systems as described herein, the present
inventors have designed and produced constructs encoding such a
PUFA PKS system and have successfully produced transgenic plants
expressing the PUFA PKS system. The transgenic plants produce oils
containing PUFAs, and the oils are substantially free of
intermediate products that accumulate in a standard PUFA pathway.
The present inventors have also demonstrated the use of the
constructs to produce PUFAs in another eukaryote, yeast, as a
proof-of-concept experiment prior to the production of the
transgenic plants. The examples demonstrate that transformation of
both yeast and plants with a PUFA PKS system that produces DHA and
DPAn-6 as the target PUFAs produces both of these PUFAs as the
primary additional fatty acids in the total fatty acids of the
plant (i.e., subtracting fatty acids that are produced in the
wild-type plant), and in the yeast and further, that any other
fatty acids that are not present in the fatty acids of the
wild-type plant or parent plant are virtually undetectable.
Specific characteristics of genetically modified plants and parts
and oils thereof of the present invention are described in detail
below.
[0325] According to the present invention, a genetically modified
plant includes a plant that has been modified using recombinant
technology, which may be combined with classical mutagenesis and
screening techniques. As used herein, genetic modifications that
result in a decrease in gene expression, in the function of the
gene, or in the function of the gene product (i.e., the protein
encoded by the gene) can be referred to as inactivation (complete
or partial), deletion, interruption, blockage or down-regulation of
a gene. For example, a genetic modification in a gene which results
in a decrease in the function of the protein encoded by such gene,
can be the result of a complete deletion of the gene (i.e., the
gene does not exist, and therefore the protein does not exist), a
mutation in the gene which results in incomplete or no translation
of the protein (e.g., the protein is not expressed), or a mutation
in the gene which decreases or abolishes the natural function of
the protein (e.g., a protein is expressed which has decreased or no
enzymatic activity or action). Genetic modifications that result in
an increase in gene expression or function can be referred to as
amplification, overproduction, overexpression, activation,
enhancement, addition, or up-regulation of a gene.
[0326] The genetic modification of a plant according to the present
invention results in the production of one or more PUFAs by the
plant. The PUFA profile and the ratio of the PUFAs produced by the
plant is not necessarily the same as the PUFA profile or ratio of
PUFAs produced by the organism from which the PUFA PKS system was
derived.
[0327] With regard to the production of genetically modified
plants, methods for the genetic engineering of plants are also well
known in the art. For instance, numerous methods for plant
transformation have been developed, including biological and
physical transformation protocols for dicotelydenous plants as well
as monocotelydenous plants (e.g. Goto-Fumiyuki et al., 1999, Nature
Biotech 17: 282-286). See, for example, Miki et al., "Procedures
for Introducing Foreign DNA into Plants" in Methods in Plant
Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.
E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 67-88. In addition,
vectors and in vitro culture methods for plant cell or tissue
transformation and regeneration of plants are available. See, for
example, Gruber et al., "Vectors for Plant Transformation" in
Methods in Plant Molecular Biology and Biotechnology, Glick, B. R.
and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp.
89-119.
[0328] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of Agrobacterium. See, for example, Horsch et
al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are
plant pathogenic soil bacteria which genetically transform plant
cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,
respectively, carry genes responsible for genetic transformation of
the plant. See, for example, Kado, C. I., Crit. Rev. Plant. Sci.
10:1 (1991). Descriptions of Agrobacterium vector systems and
methods for Agrobacterium-mediated gene transfer are provided by
numerous references, including Gruber et al., supra, Miki et al.,
supra, Moloney et al., Plant Cell Reports 8:238 (1989), and U.S.
Pat. Nos. 4,940,838 and 5,464,763.
[0329] Another generally applicable method of plant transformation
is microprojectile-mediated transformation wherein DNA is carried
on the surface of microprojectiles. The expression vector is
introduced into plant tissues with a biolistic device that
accelerates the microprojectiles to speeds sufficient to penetrate
plant cell walls and membranes. Sanford et al., Part. Sci. Technol.
5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988), Sanford,
J. C., Physiol. Plant 79:206 (1990), Klein et al., Biotechnology
10:268 (1992).
[0330] Another method for physical delivery of DNA to plants is
sonication of target cells. Zhang et al., Bio/Technology 9:996
(1991). Alternatively, liposome or spheroplast fusion have been
used to introduce expression vectors into plants. Deshayes et al.,
EMBO J., 4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. USA
84:3962 (1987). Direct uptake of DNA into protoplasts using CaCl2
precipitation, polyvinyl alcohol or poly-L-ornithine have also been
reported. Hain et al., Mol. Gen. Genet. 199:161 (1985) and Draper
et al., Plant Cell Physiol. 23:451 (1982). Electroporation of
protoplasts and whole cells and tissues have also been described.
Donn et al., In Abstracts of VIIth International Congress on Plant
Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin et
al., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol.
Biol. 24:51-61 (1994). Additionally, silicone carbide whiskers
(Kaepler et al., 1990, Plant Cell Reports) and in plant
transformation using, for example, a flower dipping methodology,
(Clough and Bent, 1998, Plant J. 16: 735-743) may be used.
[0331] The exact plant transformation methodology may vary somewhat
depending on the plant species selected and the plant cell type
selected for transformation (e.g. seedling derived cell types such
as hypocotyls and cotelydons or embryonic tissue.
[0332] As hereinbefore mentioned in one embodiment the plant
selected is safflower. A methodology to obtain safflower
transformants has been described in Baker and Dyer (Plant Cell
Reports, 1996, 16: 106-110).
[0333] Following the introduction of the genetic construct into
plant cells, plant cells are grown and upon emergence of
differentiating tissue such as shoots and roots, mature plants are
generated. Typically a plurality of plants is generated
Methodologies for regenerating plants will be generally known to
those skilled in the art and may be found in for example: Plant
Cell and Tissue Culture, 1994, Vasil and Thorpe Eds. Kluwer
Academic Publishers and in: Plant Cell Culture Protocols (Methods
in Molecular Biology 111, 1999 Hall Eds Humana Press).
[0334] Accordingly, encompassed by the present invention are
methods to genetically modify plant cells by making use of genes
from certain marine bacterial and any thraustochytrid or other
eukaryotic PUFA PKS systems, and further can utilize gene mixing to
extend and/or alter the range of PUFA products to include EPA, DHA,
DPA (n-3 or n-6), ARA, GLA, SDA and others. The method to obtain
these altered PUFA production profiles includes not only the mixing
of genes from various organisms into the thraustochytrid PUFA PKS
genes, but also various methods of genetically modifying the
endogenous thraustochytrid PUFA PKS genes disclosed herein.
Knowledge of the genetic basis and domain structure of the
thraustochytrid PUFA PKS system and the marine bacterial PUFA PKS
system provides a basis for designing novel genetically modified
organisms that produce a variety of PUFA profiles. Novel PUFA PKS
constructs prepared in microorganisms such as a thraustochytrid or
in E. coli can be isolated and used to transform plants to impart
similar PUFA production properties onto the plants. Detailed
discussions of particular modifications of PUFA PKS systems that
are encompassed by the present invention are set forth, for
example, in U.S. Patent Application Publication No. 20020194641;
U.S. Patent Application Publication No. 20040235127; and U.S.
Patent Application Publication No. 20050100995).
[0335] A genetically modified plant is preferably cultured in a
fermentation medium or grown in a suitable medium such as soil. An
appropriate, or effective, fermentation medium has been discussed
in detail above. A suitable growth medium for higher plants
includes any growth medium for plants, including, but not limited
to, soil, sand, any other particulate media that support root
growth (e.g. vermiculite, perlite, etc.) or hydroponic culture, as
well as suitable light, water and nutritional supplements which
optimize the growth of the higher plant. The genetically modified
plants of the present invention are engineered to produce PUFAs
through the activity of the PUFA PKS system. The PUFAs can be
recovered through purification processes which extract the
compounds from the plant. In a preferred embodiment, the PUFAs are
recovered by harvesting the plant. In a particularly preferred
embodiment, the PUFAs are recovered by harvesting the oil from the
plant (e.g., from the oil seeds). The plant can also be consumed in
its natural state or further processed into consumable
products.
[0336] Preferably, a genetically modified plant of the invention
produces one or more polyunsaturated fatty acids including, but not
limited to, 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), ALA (C18:3, n-3), and/or
SDA (C18:4, n-3)), and more preferably, one or more long chain
fatty acids (LCPUFAs), including, but not limited to, EPA (C20:5,
n-3), DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), or DTA (C22:4,
n-6). In a particularly preferred embodiment, a genetically
modified plant of the invention produces one or more
polyunsaturated fatty acids including, but not limited to, EPA
(C20:5, n-3), DHA (C22:6, n-3), and/or DPA (C22:5, n-6 or n-3).
[0337] Accordingly, one embodiment of the present invention relates
to a plant, and preferably an oil seed plant, wherein the plant
produces (e.g., in its mature seeds, if an oil seed plant, or in
the oil of the seeds of an oil seed plant) at least one PUFA (the
target PUFA), and wherein the total fatty acid profile in the
plant, or the part of the plant that accumulates PUFAs (e.g.,
mature seeds, if the plant is an oil seed plant or the oil of the
seeds of an oil seed plant), comprises a detectable amount of this
PUFA or PUFAs. Preferably, the target PUFA is at least a 20 carbon
PUFA and comprises at least 3 double bonds, and more preferably at
least 4 double bonds, and even more preferably, at least 5 double
bonds. Furthermore, the target PUFA is preferably a PUFA that is
not naturally produced by the plant (i.e., the wild-type plant in
the absence of genetic modification or the parent plant used as a
recipient for the indicated genetic modification). Preferably, the
total fatty acid profile in the plant or in the part of the plant
that accumulates PUFAs (including the seed oil of the plant)
comprises at least 0.1% of the target PUFA(s) by weight of the
total fatty acids, and more preferably at least about 0.2%, and
more preferably at least about 0.3%, and more preferably at least
about 0.4%, and more preferably at least about 0.5%, and more
preferably at least about 1%, and more preferably at least about
1.5%, and more preferably at least about 2%, and more preferably at
least about 2.5%, and more preferably at least about 3%, and more
preferably at least about 3.5%, and more preferably at least about
4%, and more preferably at least about 4.5%, and more preferably at
least about 5%, and more preferably at least about 5.5%, and more
preferably at least about 10%, and more preferably at least about
15%, and more preferably at least about 20%, and more preferably at
least about 25%, and more preferably at least about 30%, and more
preferably at least about 35%, and more preferably at least about
40%, and more preferably at least about 45%, and more preferably at
least about 50%, and more preferably at least about 55%, and more
preferably at least about 60%, and more preferably at least about
65%, and more preferably at least about 70%, and more preferably at
least about 75%, and more preferably more that 75% of at least one
polyunsaturated fatty acid (the target PUFA or PUFAs) by weight of
the total fatty acids produced by the plant, or any percentage from
0.1% to 75%, or greater than 75% (up to 100% or about 100%), in
0.1% increments, of the target PUFA(s). As generally used herein,
reference to a percentage amount of PUFA production is by weight of
the total fatty acids produced by the organism (plant), unless
otherwise stated (e.g., in some cases, percentage by weight is
relative to the total fatty acids produced by an enzyme complex,
such as a PUFA PKS system). In one embodiment, total fatty acids
produced by a plant are presented as a weight percent as determined
by gas chromatography (GC) analysis of a fatty acid methyl ester
(FAME) preparation, although determination of total fatty acids is
not limited to this method.
[0338] As described above, it is an additional characteristic of
the total fatty acids produced by the above-described plant (and/or
parts of plants or seed oil fraction) that these total fatty acids
produced by the plant comprise less than (or do not contain any
more than) about 10% by weight of any fatty acids, other than the
target PUFA(s) that are produced by the enzyme complex that
produces the target PUFA(s). Preferably, any fatty acids that are
produced by the enzyme complex that produces the target PUFA(s)
(e.g., as a result of genetic modification of the plant with the
enzyme or enzyme complex that produces the target PUFA(s)), other
than the target PUFA(s), are present at less than about 9%, and
more preferably less than about 8%, and more preferably less than
about 7%, and more preferably less than about 6%, and more
preferably less than about 5%, and more preferably less than about
4%, and more preferably less than about 3%, and more preferably
less than about 2%, and more preferably less than about 1% by
weight of the total fatty acids produced by the plant.
[0339] In another embodiment, any fatty acids that are produced by
the enzyme complex that produces the target PUFA(s) other than the
target PUFA(s) are present at less than (or do not contain any more
than) about 10% by weight of the total fatty acids that are
produced by the enzyme complex that produces the target PUFA(s) in
the plant (i.e., this measurement is limited to those total fatty
acids that are produced by the enzyme complex that produces the
target PUFAs), and more preferably less than about 9%, and more
preferably less than about 8%, and more preferably less than about
7%, and more preferably less than about 6%, and more preferably
less than about 5%, and more preferably less than about 4%, and
more preferably less than about 3%, and more preferably less than
about 2%, and more preferably less than about 1% by weight of the
total fatty acids, and more preferably less than about 0.5% by
weight of the total fatty acids that are produced by the enzyme
complex that produces the target PUFA(s) in the plant.
[0340] In another aspect of this embodiment of the invention, the
total fatty acids produced by the plant (and/or parts of plants or
seed oil fraction) contain less than (or do not contain any more
than) 10% PUFAs having 18 or more carbons by weight of the total
fatty acids produced by the plant, other than the target PUFA(s) or
the PUFAs that are present in the wild-type plant (not genetically
modified) or in the parent plant used as a recipient for the
indicated genetic modification. In further aspects, the total fatty
acids produced by the plant (and/or parts of plants or seed oil
fraction) contain less than 9% PUFAs having 18 or more carbons, or
less than 8% PUFAs having 18 or more carbons, or less than 7% PUFAs
having 18 or more carbons, or less than 6% PUFAs having 18 or more
carbons, or less than 5% PUFAs having 18 or more carbons, or less
than 4% PUFAs having 18 or more carbons, or less than 3% PUFAs
having 18 or more carbons, or less than 2% PUFAs having 18 or more
carbons, or less than 1% PUFAs having 18 or more carbons by weight
of the total fatty acids produced by the plant, other than the
target PUFA(s) or the PUFAs that are present in the wild-type plant
(not genetically modified) or the parent plant used as a recipient
for the indicated genetic modification.
[0341] In another aspect of this embodiment of the invention, the
total fatty acids produced by the plant (and/or parts of plants or
seed oil fraction) contain less than (or do not contain any more
than) 10% PUFAs having 20 or more carbons by weight of the total
fatty acids produced by the plant, other than the target PUFA(s) or
the PUFAs that are present in the wild-type plant (not genetically
modified) or the parent plant used as a recipient for the indicated
genetic modification. In further aspects, the total fatty acids
produced by the plant (and/or parts of plants or seed oil fraction)
contain less than 9% PUFAs having 20 or more carbons, or less than
8% PUFAs having 20 or more carbons, or less than 7% PUFAs having 20
or more carbons, or less than 6% PUFAs having 20 or more carbons,
or less than 5% PUFAs having 20 or more carbons, or less than 4%
PUFAs having 20 or more carbons, or less than 3% PUFAs having 20 or
more carbons, or less than 2% PUFAs having 20 or more carbons, or
less than 1% PUFAs having 20 or more carbons by weight of the total
fatty acids produced by the plant, other than the target PUFA(s) or
the PUFAs that are present in the wild-type plant (not genetically
modified) or the parent plant used as a recipient for the indicated
genetic modification.
[0342] In one embodiment, the total fatty acids in the plant
(and/or parts of plants or seed oil fraction) contain less than
about 10% by weight of the total fatty acids produced by the plant,
and more preferably less than about 9%, and more preferably less
than about 8%, and more preferably less than about 7%, and more
preferably less than about 6%, and more preferably less than about
5%, and more preferably less than about 4%, and more preferably
less than about 3%, and more preferably less than about 2%, and
more preferably less than about 1% of a fatty acid selected from
any one or more of: gamma-linolenic acid (GLA; 18:3, n-6);
stearidonic acid (STA or SDA; 18:4, n-3); dihomo-gamma-linolenic
acid (DGLA or HGLA; 20:3, n-6), arachidonic acid (ARA, C20:4, n-6);
eicosatrienoic acid (ETA; 20:3, n-9) and various other fatty acids,
such as 20:0; 20:1 (.DELTA.5); 20:1 (.DELTA.11); 20:2
(.DELTA.8,11); 20:2 (.DELTA.11,14); 20:3 (.DELTA.5,11,14); 20:3
(.DELTA.11,14,17); mead acid (20:3; .DELTA.5,8,11); or 20:4
(.DELTA.5,1,14,17).
[0343] In another embodiment, the fatty acids that are produced by
the enzyme system that produces the long chain PUFAs in the plant
contain less than about 10% by weight of a fatty acid selected
from: gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STA
or SDA; 18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA;
20:3, n-6), arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid
(ETA; 20:3, n-9) and various other fatty acids, such as 20:0; 20:1
(.DELTA.5); 20:1 (.DELTA.11); 20:2 (.DELTA.8,11); 20:2
(.DELTA.11,14); 20:3 (.DELTA.5,11,14); 20:3 (.DELTA.11,14,17); mead
acid (20:3; .DELTA.5,8,11); or 20:4 (.DELTA.5,1,14,17), as a
percentage of the total fatty acids produced by the plant, and more
preferably less than about 9%, and more preferably less than about
8%, and more preferably less than about 7%, and more preferably
less than about 6%, and more preferably less than about 5%, and
more preferably less than about 4%, and more preferably less than
about 3%, and more preferably less than about 2%, and more
preferably less than about 1% of a fatty acid selected from:
gamma-linolenic acid (GLA; 18:3, n-6); stearidonic acid (STA or
SDA; 18:4, n-3); dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3,
n-6), arachidonic acid (ARA, C20:4, n-6); eicosatrienoic acid (ETA;
20:3, n-9) and various other fatty acids, such as 20:0; 20:1
(.DELTA.5); 20:1 (.DELTA.11); 20:2 (.DELTA.8,11); 20:2
(.DELTA.11,14); 20:3 (.DELTA.5,11,14); 20:3 (.DELTA.11,14,17); mead
acid (20:3; .DELTA.5,8,11); or 20:4 (.DELTA.5,1,14,17).
[0344] In another embodiment, the fatty acids that are produced by
the enzyme system that produces the long chain PUFAs in the plant
contain less than about 10% by weight 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, as a
percentage of the total fatty acids produced by the plant, and more
preferably less than about 9%, and more preferably less than about
8%, and more preferably less than about 7%, and more preferably
less than about 6%, and more preferably less than about 5%, and
more preferably less than about 4%, and more preferably less than
about 3%, and more preferably less than about 2%, and more
preferably less than about 1% 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.
[0345] In another embodiment, the fatty acids that are produced by
the enzyme system that produces the long chain PUFAs in the plant
contain less than about 10% by weight 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, as a
percentage of the total fatty acids produced by the plant, and more
preferably less than about 9%, and more preferably less than about
8%, and more preferably less than about 7%, and more preferably
less than about 6%, and more preferably less than about 5%, and
more preferably less than about 4%, and more preferably less than
about 3%, and more preferably less than about 2%, and more
preferably less than about 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.
[0346] In another embodiment, the fatty acids that are produced by
the enzyme system that produces the long chain PUFAs in the plant
contain less than about 10% by weight of any one or more 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, as a
percentage of the total fatty acids produced by the plant, and more
preferably less than about 9%, and more preferably less than about
8%, and more preferably less than about 7%, and more preferably
less than about 6%, and more preferably less than about 5%, and
more preferably less than about 4%, and more preferably less than
about 3%, and more preferably less than about 2%, and more
preferably less than about 1% of any one or more 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.
[0347] In one aspect of this embodiment of the invention, the plant
produces at least two target PUFAs, and the total fatty acid
profile in the plant, or the part of the plant that accumulates
PUFAs (including oils from the oil seeds), comprises a detectable
amount of these PUFAs. In this embodiment, the PUFAs are preferably
each at least a 20 carbon PUFA and comprise at least 3 double
bonds, and more preferably at least 4 double bonds, and even more
preferably, at least 5 double bonds. Such PUFAs are most preferably
chosen from DHA, DPAn-6 and EPA. In one aspect, the plant produces
DHA and DPAn-6, and the ratio of DHA to DPAn-6 is from about 1:10
to about 10:1, including any ratio in between. In a one embodiment,
the ratio of DHA to DPA is from about 1:1 to about 3:1, and in
another embodiment, about 2.5:1. In one embodiment, the plant
produces DHA and EPA.
[0348] In another aspect of this embodiment of the invention, the
plant produces the total fatty acid profile represented by FIG. 13
or FIG. 14.
[0349] The invention further includes any seeds produced by the
plants described herein, as well as any oils produced by the plants
or seeds described herein. The invention also includes any products
produced using the plants, seed or oils described herein.
Uses for Genetically Modified Organisms of the Invention
[0350] One embodiment of the present invention is a method to
produce desired bioactive molecules (also referred to as products
or compounds) by growing or culturing a genetically modified
organism (e.g., a microorganism or a plant) of the present
invention (described in detail above). Preferably, the bioactive
molecule is a PUFA, and most preferably, an LCPUFA. Preferably, the
genetically modified organism is a genetically modified
microorganism or a genetically modified plant. Such a method
includes, for example, the step of culturing in a fermentation
medium or growing in a suitable environment, such as soil, a
microorganism or plant, respectively, that has a genetic
modification as described previously herein and in accordance with
the present invention. Preferred host cells and organisms for
genetic modification related to the PUFA PKS system of the
invention are described above.
[0351] One embodiment of the present invention is a method to
produce desired PUFAs by culturing a genetically modified
microorganism of the present invention (described in detail above).
Such a method includes the step of culturing in a fermentation
medium and under conditions effective to produce the PUFA(s) a
microorganism that has a genetic modification as described
previously herein and in accordance with the present invention. An
appropriate, or effective, medium refers to any medium in which a
genetically modified microorganism of the present invention, when
cultured, is capable of producing the desired PUFA product(s). Such
a medium is typically an aqueous medium comprising assimilable
carbon, nitrogen and phosphate sources. Such a medium can also
include appropriate salts, minerals, metals and other nutrients.
Any microorganisms of the present invention can be cultured in
conventional fermentation bioreactors. The microorganisms can be
cultured by any fermentation process which includes, but is not
limited to, batch, fed-batch, cell recycle, and continuous
fermentation. Preferred growth conditions for Thraustochytrid
microorganisms according to the present invention are well known in
the art and are described in detail, for example, in U.S. Pat. No.
5,130,242, U.S. Pat. No. 5,340,742, and U.S. Pat. No. 5,698,244,
each of which is incorporated herein by reference in its
entirety.
[0352] The desired PUFA(s) and/or other bioactive molecules
produced by the genetically modified microorganism can be recovered
from the fermentation medium using conventional separation and
purification techniques. For example, the fermentation medium can
be filtered or centrifuged to remove microorganisms, cell debris
and other particulate matter, and the product can be recovered from
the cell-free supernatant by conventional methods, such as, for
example, ion exchange, chromatography, extraction, solvent
extraction, phase separation, membrane separation, electrodialysis,
reverse osmosis, distillation, chemical derivatization and
crystallization. Alternatively, microorganisms producing the
PUFA(s), or extracts and various fractions thereof, can be used
without removal of the microorganism components from the
product.
[0353] Preferably, PUFAs are produced in an amount that is greater
than about 5% of the dry weight of the microorganism, and in one
aspect, in an amount that is greater than 6%, and in another
aspect, in an amount that is greater than 7%, and in another
aspect, in an amount that is greater than 8%, and in another
aspect, in an amount that is greater than 9%, and in another
aspect, in an amount that is greater than 10%, and so on in whole
integer percentages, up to greater than 90% dry weight of the
microorganism (e.g., 15%, 20%, 30%, 40%, 50%, and any percentage in
between).
[0354] Preferably, bioactive compounds of interest are produced by
the genetically modified microorganism in an amount that is greater
than about 0.05%, and preferably greater than about 0.1%, and more
preferably greater than about 0.25%, and more preferably greater
than about 0.5%, and more preferably greater than about 0.75%, and
more preferably greater than about 1%, and more preferably greater
than about 2.5%, and more preferably greater than about 5%, and
more preferably greater than about 10%, and more preferably greater
than about 15%, and even more preferably greater than about 20% of
the dry weight of the microorganism. For lipid compounds,
preferably, such compounds are produced in an amount that is
greater than about 5% of the dry weight of the microorganism. For
other bioactive compounds, such as antibiotics or compounds that
are synthesized in smaller amounts, those strains possessing such
compounds at of the dry weight of the microorganism are identified
as predictably containing a novel PKS system of the type described
above. In some embodiments, particular bioactive molecules
(compounds) are secreted by the microorganism, rather than
accumulating. Therefore, such bioactive molecules are generally
recovered from the culture medium and the concentration of molecule
produced will vary depending on the microorganism and the size of
the culture.
[0355] In the method for production of desired bioactive compounds
of the present invention, a genetically modified plant is cultured
in a fermentation medium or grown in a suitable medium such as
soil. An appropriate, or effective, fermentation medium has been
discussed in detail above. A suitable growth medium for higher
plants includes any growth medium for plants, including, but not
limited to, soil, sand, any other particulate media that support
root growth (e.g. vermiculite, perlite, etc.) or hydroponic
culture, as well as suitable light, water and nutritional
supplements which optimize the growth of the higher plant. The
genetically modified plants of the present invention are engineered
to produce significant quantities of the desired product through
the activity of the PUFA PKS system and other heterologous proteins
(accessory proteins to the PUFA PKS system) according to the
present invention. The compounds can be recovered through
purification processes which extract the compounds from the plant.
In a preferred embodiment, the compound is recovered by harvesting
the plant. In this embodiment, the plant can be consumed in its
natural state or further processed into consumable products.
[0356] The invention further includes any organisms or parts
thereof described herein (e.g., microorganisms and preparations or
fractions thereof or plants, parts of the plants (e.g., oil seeds),
or preparations or fractions thereof), as well as any oils produced
by the organisms described herein. The invention also includes any
products produced using the organisms, parts thereof, or oils
described herein.
[0357] One embodiment of the present invention relates to a method
to modify a product containing at least one fatty acid, comprising
adding to the product an organism, part thereof, or oil produced by
a genetically modified organism according to the invention and as
described herein (e.g., a plant or microorganism that has been
genetically modified with a PUFA PKS system, makes use of any of
the strategies for improvement of production and/or accumulation of
PUFAs described herein, and has a fatty acid profile described
herein). Any products produced by this method or generally
containing any organisms, parts thereof, or oils from the organisms
described herein are also encompassed by the invention.
[0358] Preferably, the product is selected from the group
consisting of a food, a dietary supplement, a pharmaceutical
formulation, a humanized animal milk, and an infant formula.
Suitable pharmaceutical formulations include, but are not limited
to, an anti-inflammatory formulation, a chemotherapeutic agent, an
active excipient, an osteoporosis drug, an anti-depressant, an
anti-convulsant, an anti-Heliobactor pylori drug, a drug for
treatment of neurodegenerative disease, a drug for treatment of
degenerative liver disease, an antibiotic, and a cholesterol
lowering formulation. In one embodiment, the product is used to
treat a condition selected from the group consisting of: chronic
inflammation, acute inflammation, gastrointestinal disorder,
cancer, cachexia, cardiac restenosis, neurodegenerative disorder,
degenerative disorder of the liver, blood lipid disorder,
osteoporosis, osteoarthritis, autoimmune disease, preeclampsia,
preterm birth, age related maculopathy, pulmonary disorder, and
peroxisomal disorder.
[0359] Suitable food products include, but are not limited to, fine
bakery wares, bread and rolls, breakfast cereals, processed and
unprocessed cheese, condiments (ketchup, mayonnaise, etc.), dairy
products (milk, yogurt), puddings and gelatine desserts, carbonated
drinks, teas, powdered beverage mixes, processed fish products,
fruit-based drinks, chewing gum, hard confectionery, frozen dairy
products, processed meat products, nut and nut-based spreads,
pasta, processed poultry products, gravies and sauces, potato chips
and other chips or crisps, chocolate and other confectionery, soups
and soup mixes, soya based products (milks, drinks, creams,
whiteners), vegetable oil-based spreads, and vegetable-based
drinks.
General Definitions and Guidance
[0360] According to the present invention, an isolated protein is a
protein or a fragment thereof (including a polypeptide or peptide)
that has been removed from its natural milieu (i.e., that has been
subject to human manipulation) and can include purified proteins,
partially purified proteins, recombinantly produced proteins, and
synthetically produced proteins, for example. As such, "isolated"
does not reflect the extent to which the protein has been purified.
Preferably, an isolated protein of the present invention is
produced recombinantly. An isolated peptide can be produced
synthetically (e.g., chemically, such as by peptide synthesis) or
recombinantly.
[0361] As used herein, the term "lipid" includes phospholipids;
free fatty acids; esters of fatty acids; triacylglycerols;
diacylglycerides; monoacylglycerides; lysophospholipids; soaps;
phosphatides; waxes (esters of alcohols and fatty acids); sterols
and sterol esters; carotenoids; xanthophylls (e.g.,
oxycarotenoids); hydrocarbons; and other lipids known to one of
ordinary skill in the art. The terms "polyunsaturated fatty acid"
and "PUFA" include not only the free fatty acid form, but other
forms as well, such as the TAG form and the PL form.
[0362] Reference to a particular protein from a specific organism
or to a particular protein being derived from a specific organism,
such as a "Schizochytrium ACoAS" or an "ACoAS derived from
Schizochytrium", by way of example, refers to an ACoAS (including a
homologue of the naturally occurring ACoAS) from a Schizochytrium
or an ACoAS that has been otherwise produced from the knowledge of
the structure (e.g., sequence) of a naturally occurring ACoAS from
Schizochytrium. In other words, a Schizochytrium ACoAS includes any
ACoAS that has the structure and function of a naturally occurring
ACoAS from Schizochytrium or that has a structure and function that
is sufficiently similar to a Schizochytrium ACoAS such that the
ACoAS is a biologically active (i.e., has biological activity)
homologue of a naturally occurring ACoAS from Schizochytrium. As
such, a Schizochytrium ACoAS can include purified, partially
purified, recombinant, mutated/modified and synthetic proteins.
[0363] According to the present invention, the terms "modification"
and "mutation" can be used interchangeably, particularly with
regard to the modifications/mutations to the primary amino acid
sequences of a protein or peptide (or nucleic acid sequences)
described herein. The term "modification" can also be used to
describe post-translational modifications to a protein or peptide
including, but not limited to, methylation, farnesylation,
carboxymethylation, geranyl geranylation, glycosylation,
phosphorylation, acetylation, myristoylation, prenylation,
palmitation, and/or amidation. Modifications can also include, for
example, complexing a protein or peptide with another compound.
Such modifications can be considered to be mutations, for example,
if the modification is different than the post-translational
modification that occurs in the natural, wild-type protein or
peptide.
[0364] As used herein, the term "homologue" is used to refer to a
protein or peptide which differs from a naturally occurring protein
or peptide (i.e., the "prototype" or "wild-type" protein) by one or
more minor modifications or mutations to the naturally occurring
protein or peptide, but which maintains the overall basic protein
and side chain structure of the naturally occurring form (i.e.,
such that the homologue is identifiable as being related to the
wild-type protein). Such changes include, but are not limited to:
changes in one or a few (e.g., 1% or less) amino acid side chains;
changes one or a few (e.g., 1% or less) amino acids, including
deletions (e.g., a truncated version of the protein or peptide)
insertions and/or substitutions; changes in stereochemistry of one
or a few (e.g., 1% or less) atoms; and/or minor derivatizations,
including but not limited to: methylation, farnesylation, geranyl
geranylation, glycosylation, carboxymethylation, phosphorylation,
acetylation, myristoylation, prenylation, palmitation, and/or
amidation. A homologue can have either enhanced, decreased, or
substantially similar properties as compared to the naturally
occurring protein or peptide. Preferred homologues of a protein are
described in detail below. It is noted that homologues can include
synthetically produced homologues, naturally occurring allelic
variants of a given protein or domain thereof, or homologous
sequences from organisms other than the organism from which the
reference sequence was derived.
[0365] Conservative substitutions typically include substitutions
within the following groups: glycine and alanine; valine,
isoleucine and leucine; aspartic acid, glutamic acid, asparagine,
and glutamine; serine and threonine; lysine and arginine; and
phenylalanine and tyrosine. Substitutions may also be made on the
basis of conserved hydrophobicity or hydrophilicity (Kyte and
Doolittle, J. Mol. Biol. 157:105 (1982)), or on the basis of the
ability to assume similar polypeptide secondary structure (Chou and
Fasman, Adv. Enzymol. 47: 45 (1978)).
[0366] Homologues can be the result of natural allelic variation or
natural mutation. A naturally occurring allelic variant of a
nucleic acid encoding a protein is a gene that occurs at
essentially the same locus (or loci) in the genome as the gene
which encodes such protein, but which, due to natural variations
caused by, for example, mutation or recombination, has a similar
but not identical sequence. Allelic variants typically encode
proteins having similar activity to that of the protein encoded by
the gene to which they are being compared. One class of allelic
variants can encode the same protein but have different nucleic
acid sequences due to the degeneracy of the genetic code. Allelic
variants can also comprise alterations in the 5' or 3' untranslated
regions of the gene (e.g., in regulatory control regions). Allelic
variants are well known to those skilled in the art.
[0367] Homologues can be produced using techniques known in the art
for the production of proteins including, but not limited to,
direct modifications to the isolated, naturally occurring protein,
direct protein synthesis, or modifications to the nucleic acid
sequence encoding the protein using, for example, classic or
recombinant DNA techniques to effect random or targeted
mutagenesis.
[0368] Modifications or mutations in protein homologues, as
compared to the wild-type protein, either increase, decrease, or do
not substantially change, the basic biological activity of the
homologue as compared to the naturally occurring (wild-type)
protein. In general, the biological activity or biological action
of a protein refers to any function(s) exhibited or performed by
the protein that is ascribed to the naturally occurring form of the
protein as measured or observed in vivo (i.e., in the natural
physiological environment of the protein) or in vitro (i.e., under
laboratory conditions). Biological activities of PUFA PKS systems
and the individual proteins/domains that make up a PUFA PKS system
have been described in detail elsewhere herein and in the
referenced patents and applications. Biological activities of an
ACoAS include binding to a substrate, and preferably for the
present invention, a free fatty acid (FFA) of a PUFA, and
catalyzing the conversion of the FFA to an acyl-CoA PUFA.
[0369] Modifications of a protein, such as in a homologue, may
result in proteins having the same biological activity as the
naturally occurring protein, or in proteins having decreased or
increased biological activity as compared to the naturally
occurring protein. Modifications which result in a decrease in
protein expression or a decrease in the activity of the protein,
can be referred to as inactivation (complete or partial),
down-regulation, or decreased action (or activity) of a protein.
Similarly, modifications which result in an increase in protein
expression or an increase in the activity of the protein, can be
referred to as amplification, overproduction, activation,
enhancement, up-regulation or increased action (or activity) of a
protein. It is noted that general reference to a homologue having
the biological activity of the wild-type protein does not
necessarily mean that the homologue has identical biological
activity as the wild-type protein, particularly with regard to the
level of biological activity. Rather, a homologue can perform the
same biological activity as the wild-type protein, but at a reduced
or increased level of activity as compared to the wild-type
protein. A functional domain of a protein is a domain (i.e., a
domain can be a portion of a protein) that is capable of performing
a biological function (i.e., has biological activity).
[0370] Methods of detecting a protein or measuring the activity of
a protein include, but are not limited to, measurement of
transcription of the protein, measurement of translation of the
protein, measurement of posttranslational modification of the
protein, measurement of enzymatic activity of the protein, and/or
measurement of production of one or more products resulting from
the activity of the protein (e.g., PUFA production). It is noted
that an isolated protein of the present invention (including a
homologue) is not necessarily required to have the biological
activity of the wild-type protein. For example, a protein can be a
truncated, mutated or inactive protein, for example. Such proteins
are useful in screening assays, for example, or for other purposes
such as antibody production. In a preferred embodiment, the
isolated proteins of the present invention have a biological
activity that is similar to that of the wild-type protein (although
not necessarily equivalent, as discussed above).
[0371] Methods to measure protein expression levels generally
include, but are not limited to: Western blot, immunoblot,
enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA),
immunoprecipitation, surface plasmon resonance, chemiluminescence,
fluorescent polarization, phosphorescence, immunohistochemical
analysis, matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry, microcytometry,
microarray, microscopy, fluorescence activated cell sorting (FACS),
and flow cytometry, as well as assays based on a property of the
protein including but not limited to enzymatic activity or
interaction with other protein partners. Binding assays are also
well known in the art. For example, a BIAcore machine can be used
to determine the binding constant of a complex between two
proteins. The dissociation constant for the complex can be
determined by monitoring changes in the refractive index with
respect to time as buffer is passed over the chip (O'Shannessy et
al. Anal. Biochem. 212:457 (1993); Schuster et al., Nature 365:343
(1993)). Other suitable assays for measuring the binding of one
protein to another include, for example, immunoassays such as
enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays
(RIA); or determination of binding by monitoring the change in the
spectroscopic or optical properties of the proteins through
fluorescence, UV absorption, circular dichroism, or nuclear
magnetic resonance (NMR).
[0372] In one aspect of the invention, a protein encompassed by the
present invention, including a homologue of a particular protein
described herein, comprises an amino acid sequence that includes at
least about 100 consecutive amino acids of the amino acid sequence
from the reference protein, wherein the amino acid sequence of the
homologue has a biological activity of the protein as described
herein. In a further aspect, the amino acid sequence of the protein
is comprises at least about 200 consecutive amino acids, and more
preferably at least about 300 consecutive amino acids, and more
preferably at least about 400 consecutive amino acids, and can
include 500 consecutive amino acids, or more of the amino acid
sequence of the reference protein, up to the full-length of the
protein, including any increment that is a whole number integer
(e.g., 200, 201, 202, 203, etc.).
[0373] According to the present invention, the term "contiguous" or
"consecutive", with regard to nucleic acid or amino acid sequences
described herein, means to be connected in an unbroken sequence.
For example, for a first sequence to comprise 30 contiguous (or
consecutive) amino acids of a second sequence, means that the first
sequence includes an unbroken sequence of 30 amino acid residues
that is 100% identical to an unbroken sequence of 30 amino acid
residues in the second sequence. Similarly, for a first sequence to
have "100% identity" with a second sequence means that the first
sequence exactly matches the second sequence with no gaps between
nucleotides or amino acids.
[0374] Typically, a homologue of a reference protein, such as any
of the ACoAS proteins described herein, has an amino acid sequence
that is at least about 50% identical, and more preferably at least
about 55% identical, and more preferably at least about 60%
identical, and more preferably at least about 65% identical, and
more preferably at least about 70% identical, and more preferably
at least about 75% identical, and more preferably at least about
80% identical, and more preferably at least about 85% identical,
and more preferably at least about 90% identical, and more
preferably at least about 95% identical, and more preferably at
least about 96% identical, and more preferably at least about 97%
identical, and more preferably at least about 98% identical, and
more preferably at least about 99% identical (or any percentage
between 60% and 99%, in whole single percentage increments) to the
amino acid sequence of the reference protein (e.g., to an ACoAS
protein). The homologue preferably has a biological activity of the
protein or domain from which it is derived or related (i.e., the
protein or domain having the reference amino acid sequence). With
regard to ACoAS homologues, the homologue preferably has ACoAS
enzymatic activity, and more specifically, the ability to catalyze
the conversion of long chain PUFA free fatty acids (FFA) to
acyl-CoA. With regard to other accessory proteins described herein,
such proteins can have the biological activity of, for example,
utilizing PUFA-CoA as substrates in forming PL or TAG.
[0375] As used herein, unless otherwise specified, reference to a
percent (%) identity refers to an evaluation of homology which is
performed using: (1) a BLAST 2.0 Basic BLAST homology search using
blastp for amino acid searches, blastn for nucleic acid searches,
and blastX for nucleic acid searches and searches of translated
amino acids in all 6 open reading frames, all with standard default
parameters, wherein the query sequence is filtered for low
complexity regions by default (described in Altschul, S. F.,
Madden, T. L., Schaaffer, A. A., Zhang, J., Zhang, Z., Miller, W.
& Lipman, D. J. (1997) "Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs." Nucleic Acids Res.
25:3389, incorporated herein by reference in its entirety); (2) a
BLAST 2 alignment (using the parameters described below); (3)
and/or PSI-BLAST with the standard default parameters
(Position-Specific Iterated BLAST). It is noted that due to some
differences in the standard parameters between BLAST 2.0 Basic
BLAST and BLAST 2, two specific sequences might be recognized as
having significant homology using the BLAST 2 program, whereas a
search performed in BLAST 2.0 Basic BLAST using one of the
sequences as the query sequence may not identify the second
sequence in the top matches. In addition, PSI-BLAST provides an
automated, easy-to-use version of a "profile" search, which is a
sensitive way to look for sequence homologues. The program first
performs a gapped BLAST database search. The PSI-BLAST program uses
the information from any significant alignments returned to
construct a position-specific score matrix, which replaces the
query sequence for the next round of database searching. Therefore,
it is to be understood that percent identity can be determined by
using any one of these programs.
[0376] Two specific sequences can be aligned to one another using
BLAST 2 sequence as described in Tatusova and Madden, "Blast 2
sequences--a new tool for comparing protein and nucleotide
sequences", FEMS Microbiol Lett. 174:247 (1999), incorporated
herein by reference in its entirety. BLAST 2 sequence alignment is
performed in blastp or blastn using the BLAST 2.0 algorithm to
perform a Gapped BLAST search (BLAST 2.0) between the two sequences
allowing for the introduction of gaps (deletions and insertions) in
the resulting alignment. For purposes of clarity herein, a BLAST 2
sequence alignment is performed using the standard default
parameters as follows.
For blastn, using 0 BLOSUM62 matrix:
[0377] Reward for match=1
[0378] Penalty for mismatch=-2
[0379] Open gap (5) and extension gap (2) penalties
[0380] gap x_dropoff (50) expect (10) word size (11) filter
(on)
[0381] For blastp, using 0 BLOSUM62 matrix:
[0382] Open gap (11) and extension gap (1) penalties
[0383] gap x_dropoff (50) expect (10) word size (3) filter
(on).
[0384] In one embodiment of the present invention, an isolated
protein or domain of the present invention comprises, consists
essentially of, or consists of, any of the amino acid sequences
described in any of U.S. Pat. No. 6,566,583; Metz et al., Science
293:290-293 (2001); U.S. Patent Application Publication No.
20020194641; U.S. Patent Application Publication No. 20040235127;
U.S. Patent Application Publication No. 20050100995; and U.S.
Provisional Application No. 60/689,167, filed Jun. 10, 2005, or any
biologically active fragments or domains thereof. These proteins
are proteins of the PUFA PKS system and can be used in connection
with any of the accessory proteins described herein.
[0385] In another embodiment of the invention, an amino acid
sequence having the biological activity of a protein described
herein (e.g., an ACoAS protein) includes an amino acid sequence
that is sufficiently similar to the naturally occurring protein or
polypeptide that is specifically described herein that a nucleic
acid sequence encoding the amino acid sequence is capable of
hybridizing under moderate, high, or very high stringency
conditions (described below) to (i.e., with) a nucleic acid
molecule encoding the naturally occurring protein or polypeptide
(i.e., to the complement of the nucleic acid strand encoding the
naturally occurring protein or polypeptide). Preferably, an amino
acid sequence having the biological activity of a protein described
herein is encoded by a nucleic acid sequence that hybridizes under
moderate, high or very high stringency conditions to the complement
of a nucleic acid sequence that encodes any of the amino acid
sequences described herein. Methods to deduce a complementary
sequence are known to those skilled in the art. It should be noted
that since amino acid sequencing and nucleic acid sequencing
technologies are not entirely error-free, the sequences presented
herein, at best, represent apparent sequences of the proteins
encompassed by the present invention.
[0386] As used herein, hybridization conditions refer to standard
hybridization conditions under which nucleic acid molecules are
used to identify similar nucleic acid molecules. Such standard
conditions are disclosed, for example, in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs
Press (1989). Sambrook et al., ibid., is incorporated by reference
herein in its entirety (see specifically, pages 9.31-9.62). In
addition, formulae to calculate the appropriate hybridization and
wash conditions to achieve hybridization permitting varying degrees
of mismatch of nucleotides are disclosed, for example, in Meinkoth
et al., Anal. Biochem. 138, 267 (1984); Meinkoth et al., ibid., is
incorporated by reference herein in its entirety.
[0387] More particularly, moderate stringency hybridization and
washing conditions, as referred to herein, refer to conditions
which permit isolation of nucleic acid molecules having at least
about 70% nucleic acid sequence identity with the nucleic acid
molecule being used to probe in the hybridization reaction (i.e.,
conditions permitting about 30% or less mismatch of nucleotides).
High stringency hybridization and washing conditions, as referred
to herein, refer to conditions which permit isolation of nucleic
acid molecules having at least about 80% nucleic acid sequence
identity with the nucleic acid molecule being used to probe in the
hybridization reaction (i.e., conditions permitting about 20% or
less mismatch of nucleotides). Very high stringency hybridization
and washing conditions, as referred to herein, refer to conditions
which permit isolation of nucleic acid molecules having at least
about 90% nucleic acid sequence identity with the nucleic acid
molecule being used to probe in the hybridization reaction (i.e.,
conditions permitting about 10% or less mismatch of nucleotides).
As discussed above, one of skill in the art can use the formulae in
Meinkoth et al., ibid. to calculate the appropriate hybridization
and wash conditions to achieve these particular levels of
nucleotide mismatch. Such conditions will vary, depending on
whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated
melting temperatures for DNA:DNA hybrids are 10.degree. C. less
than for DNA:RNA hybrids. In particular embodiments, stringent
hybridization conditions for DNA:DNA hybrids include hybridization
at an ionic strength of 6.times.SSC (0.9 M Na.sup.+) at a
temperature of between about 20.degree. C. and about 35.degree. C.
(lower stringency), more preferably, between about 28.degree. C.
and about 40.degree. C. (more stringent), and even more preferably,
between about 35.degree. C. and about 45.degree. C. (even more
stringent), with appropriate wash conditions. In particular
embodiments, stringent hybridization conditions for DNA:RNA hybrids
include hybridization at an ionic strength of 6.times.SSC (0.9 M
Na.sup.+) at a temperature of between about 30.degree. C. and about
45.degree. C., more preferably, between about 38.degree. C. and
about 50.degree. C., and even more preferably, between about
45.degree. C. and about 55.degree. C., with similarly stringent
wash conditions. These values are based on calculations of a
melting temperature for molecules larger than about 100
nucleotides, 0% formamide and a G+C content of about 40%.
Alternatively, T.sub.m can be calculated empirically as set forth
in Sambrook et al., supra, pages 9.31 to 9.62. In general, the wash
conditions should be as stringent as possible, and should be
appropriate for the chosen hybridization conditions. For example,
hybridization conditions can include a combination of salt and
temperature conditions that are approximately 20-25.degree. C.
below the calculated T.sub.m of a particular hybrid, and wash
conditions typically include a combination of salt and temperature
conditions that are approximately 12-20.degree. C. below the
calculated T.sub.m of the particular hybrid. One example of
hybridization conditions suitable for use with DNA:DNA hybrids
includes a 2-24 hour hybridization in 6.times.SSC (50% formamide)
at about 42.degree. C., followed by washing steps that include one
or more washes at room temperature in about 2.times.SSC, followed
by additional washes at higher temperatures and lower ionic
strength (e.g., at least one wash as about 37.degree. C. in about
0.1.times.-0.5.times.SSC, followed by at least one wash at about
68.degree. C. in about 0.1.times.-0.5.times.SSC).
[0388] The present invention also includes a fusion protein that
includes any protein or any homologue or fragment thereof of the
present invention attached to one or more fusion segments. Suitable
fusion segments for use with the present invention include, but are
not limited to, segments that can: enhance a protein's stability;
provide other desirable biological activity; and/or assist with the
purification of the protein (e.g., by affinity chromatography). A
suitable fusion segment can be a domain of any size that has the
desired function (e.g., imparts increased stability, solubility,
biological activity; and/or simplifies purification of a protein).
Fusion segments can be joined to amino and/or carboxyl termini of
the protein and can be susceptible to cleavage in order to enable
straight-forward recovery of the desired protein. Fusion proteins
are preferably produced by culturing a recombinant cell transfected
with a fusion nucleic acid molecule that encodes a protein
including the fusion segment attached to either the carboxyl and/or
amino terminal end of the protein of the invention as discussed
above.
[0389] In one embodiment of the present invention, any of the amino
acid sequences described herein, as well as homologues of such
sequences, can be produced with from at least one, and up to about
20, additional heterologous amino acids flanking each of the C-
and/or N-terminal end of the given amino acid sequence. The
resulting protein or polypeptide can be referred to as "consisting
essentially of" a given amino acid sequence. According to the
present invention, the heterologous amino acids are a sequence of
amino acids that are not naturally found (i.e., not found in
nature, in vivo) flanking the given amino acid sequence or which
would not be encoded by the nucleotides that flank the naturally
occurring nucleic acid sequence encoding the given amino acid
sequence as it occurs in the gene, if such nucleotides in the
naturally occurring sequence were translated using standard codon
usage for the organism from which the given amino acid sequence is
derived. Similarly, the phrase "consisting essentially of", when
used with reference to a nucleic acid sequence herein, refers to a
nucleic acid sequence encoding a given amino acid sequence that can
be flanked by from at least one, and up to as many as about 60,
additional heterologous nucleotides at each of the 5' and/or the 3'
end of the nucleic acid sequence encoding the given amino acid
sequence. The heterologous nucleotides are not naturally found
(i.e., not found in nature, in vivo) flanking the nucleic acid
sequence encoding the given amino acid sequence as it occurs in the
natural gene.
[0390] The minimum size of a protein or domain and/or a homologue
or fragment thereof of the present invention is, in one aspect, a
size sufficient to have the requisite biological activity, or
sufficient to serve as an antigen for the generation of an antibody
or as a target in an in vitro assay. In one embodiment, a protein
of the present invention is at least about 8 amino acids in length
(e.g., suitable for an antibody epitope or as a detectable peptide
in an assay), or at least about 25 amino acids in length, or at
least about 50 amino acids in length, or at least about 100 amino
acids in length, or at least about 150 amino acids in length, or at
least about 200 amino acids in length, or at least about 250 amino
acids in length, or at least about 300 amino acids in length, or at
least about 350 amino acids in length, or at least about 400 amino
acids in length, or at least about 450 amino acids in length, or at
least about 500 amino acids in length, and so on, in any length
between 8 amino acids and up to the full length of a protein or
domain of the invention or longer, in whole integers (e.g., 8, 9,
10, . . . 25, 26, . . . 500, 501, . . . ). There is no limit, other
than a practical limit, on the maximum size of such a protein in
that the protein can include a portion of the protein, domain, or
biologically active or useful fragment thereof, or a full-length
protein or domain, plus additional sequence (e.g., a fusion protein
sequence), if desired.
[0391] Another embodiment of the present invention relates to
isolated nucleic acid molecules comprising, consisting essentially
of, or consisting of nucleic acid sequences that encode any of the
proteins described herein, including a homologue or fragment of any
of such proteins, as well as nucleic acid sequences that are fully
complementary thereto. In accordance with the present invention, an
isolated nucleic acid molecule is a nucleic acid molecule that has
been removed from its natural milieu (i.e., that has been subject
to human manipulation), its natural milieu being the genome or
chromosome in which the nucleic acid molecule is found in nature.
As such, "isolated" does not necessarily reflect the extent to
which the nucleic acid molecule has been purified, but indicates
that the molecule does not include an entire genome or an entire
chromosome in which the nucleic acid molecule is found in nature.
An isolated nucleic acid molecule can include a gene. An isolated
nucleic acid molecule that includes a gene is not a fragment of a
chromosome that includes such gene, but rather includes the coding
region and regulatory regions associated with the gene, but no
additional genes that are naturally found on the same chromosome,
with the exception of other genes that encode other proteins of the
PUFA PKS system as described herein, when the nucleic acid molecule
encodes a core PUFA PKS protein. An isolated nucleic acid molecule
can also include a specified nucleic acid sequence flanked by
(i.e., at the 5' and/or the 3' end of the sequence) additional
nucleic acids that do not normally flank the specified nucleic acid
sequence in nature (i.e., heterologous sequences). Isolated nucleic
acid molecule can include DNA, RNA (e.g., mRNA), or derivatives of
either DNA or RNA (e.g., cDNA). Although the phrase "nucleic acid
molecule" primarily refers to the physical nucleic acid molecule
and the phrase "nucleic acid sequence" primarily refers to the
sequence of nucleotides on the nucleic acid molecule, the two
phrases can be used interchangeably, especially with respect to a
nucleic acid molecule, or a nucleic acid sequence, being capable of
encoding a protein or a domain of a protein.
[0392] Preferably, an isolated nucleic acid molecule of the present
invention is produced using recombinant DNA technology (e.g.,
polymerase chain reaction (PCR) amplification, cloning) or chemical
synthesis. Isolated nucleic acid molecules include natural nucleic
acid molecules and homologues thereof, including, but not limited
to, natural allelic variants and modified nucleic acid molecules in
which nucleotides have been inserted, deleted, substituted, and/or
inverted in such a manner that such modifications provide the
desired effect (e.g., retain, improve or decrease activity of the
protein). Protein homologues (e.g., proteins encoded by nucleic
acid homologues) have been discussed in detail above.
[0393] A nucleic acid molecule homologue can be produced using a
number of methods known to those skilled in the art (see, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Labs Press (1989)). For example, nucleic acid
molecules can be modified using a variety of techniques including,
but not limited to, classic mutagenesis techniques and recombinant
DNA techniques, such as site-directed mutagenesis, chemical
treatment of a nucleic acid molecule to induce mutations,
restriction enzyme cleavage of a nucleic acid fragment, ligation of
nucleic acid fragments, PCR amplification and/or mutagenesis of
selected regions of a nucleic acid sequence, synthesis of
oligonucleotide mixtures and ligation of mixture groups to "build"
a mixture of nucleic acid molecules and combinations thereof.
Nucleic acid molecule homologues can be selected from a mixture of
modified nucleic acids by screening for the function of the protein
encoded by the nucleic acid and/or by hybridization with a
wild-type gene.
[0394] The minimum size of a nucleic acid molecule of the present
invention is a size sufficient to form a probe or oligonucleotide
primer that is capable of forming a stable hybrid (e.g., under
moderate, high or very high stringency conditions) with the
complementary sequence of a nucleic acid molecule of the present
invention, or of a size sufficient to encode an amino acid sequence
having a biological activity of a protein according to the present
invention. As such, the size of the nucleic acid molecule encoding
such a protein can be dependent on the nucleic acid composition and
percent homology or identity between the nucleic acid molecule and
complementary sequence as well as upon hybridization conditions per
se (e.g., temperature, salt concentration, and formamide
concentration). The minimal size of a nucleic acid molecule that is
used as an oligonucleotide primer or as a probe is typically at
least about 12 to about 15 nucleotides in length if the nucleic
acid molecules are GC-rich and at least about 15 to about 18 bases
in length if they are AT-rich. There is no limit, other than a
practical limit, on the maximal size of a nucleic acid molecule of
the present invention, in that the nucleic acid molecule can
include a sequence sufficient to encode a biologically active
fragment of a protein or the full-length protein.
[0395] Another embodiment of the present invention includes a
recombinant nucleic acid molecule comprising a recombinant vector
and a nucleic acid sequence encoding a protein or peptide having a
biological activity of any of the proteins described herein. Such
nucleic acid sequences are described in detail above. According to
the present invention, a recombinant vector is an engineered (i.e.,
artificially produced) nucleic acid molecule that is used as a tool
for manipulating a nucleic acid sequence of choice and for
introducing such a nucleic acid sequence into a host cell. The
recombinant vector is therefore suitable for use in cloning,
sequencing, and/or otherwise manipulating the nucleic acid sequence
of choice, such as by expressing and/or delivering the nucleic acid
sequence of choice into a host cell to form a recombinant cell.
Such a vector typically contains heterologous nucleic acid
sequences, that is nucleic acid sequences that are not naturally
found adjacent to nucleic acid sequence to be cloned or delivered,
although the vector can also contain regulatory nucleic acid
sequences (e.g., promoters, untranslated regions) which are
naturally found adjacent to nucleic acid molecules of the present
invention or which are useful for expression of the nucleic acid
molecules of the present invention (discussed in detail below). The
vector can be either RNA or DNA, either prokaryotic or eukaryotic,
and typically is a plasmid. The vector can be maintained as an
extrachromosomal element (e.g., a plasmid) or it can be integrated
into the chromosome of a recombinant organism (e.g., a microbe or a
plant). The entire vector can remain in place within a host cell,
or under certain conditions, the plasmid DNA can be deleted,
leaving behind the nucleic acid molecule of the present invention.
The integrated nucleic acid molecule can be under chromosomal
promoter control, under native or plasmid promoter control, or
under a combination of several promoter controls. Single or
multiple copies of the nucleic acid molecule can be integrated into
the chromosome. A recombinant vector of the present invention can
contain at least one selectable marker.
[0396] In one embodiment, a recombinant vector used in a
recombinant nucleic acid molecule of the present invention is an
expression vector. As used herein, the phrase "expression vector"
is used to refer to a vector that is suitable for production of an
encoded product (e.g., a protein of interest). In this embodiment,
a nucleic acid sequence encoding the product to be produced (e.g.,
a PUFA PKS domain or protein) is inserted into the recombinant
vector to produce a recombinant nucleic acid molecule. The nucleic
acid sequence encoding the protein to be produced is inserted into
the vector in a manner that operatively links the nucleic acid
sequence to regulatory sequences in the vector that enable the
transcription and translation of the nucleic acid sequence within
the recombinant host cell.
[0397] In another embodiment, a recombinant vector used in a
recombinant nucleic acid molecule of the present invention is a
targeting vector. As used herein, the phrase "targeting vector" is
used to refer to a vector that is used to deliver a particular
nucleic acid molecule into a recombinant host cell, wherein the
nucleic acid molecule is used to delete, inactivate, or replace an
endogenous gene or portion of a gene within the host cell or
microorganism (i.e., used for targeted gene disruption or knock-out
technology). Such a vector may also be known in the art as a
"knock-out" vector. In one aspect of this embodiment, a portion of
the vector, but more typically, the nucleic acid molecule inserted
into the vector (i.e., the insert), has a nucleic acid sequence
that is homologous to a nucleic acid sequence of a target gene in
the host cell (i.e., a gene which is targeted to be deleted or
inactivated). The nucleic acid sequence of the vector insert is
designed to associate with the target gene such that the target
gene and the insert may undergo homologous recombination, whereby
the endogenous target gene is deleted, inactivated, attenuated
(i.e., by at least a portion of the endogenous target gene being
mutated or deleted), or replaced. The use of this type of
recombinant vector to replace an endogenous Schizochytrium gene,
for example, with a recombinant gene has been previously described
by the present inventors, and the general technique for genetic
transformation of Thraustochytrids is described in detail in U.S.
patent application Ser. No. 10/124,807, published as U.S. Patent
Application Publication No. 20030166207, published Sep. 4, 2003.
Genetic transformation techniques for plants are well-known in the
art.
[0398] Typically, a recombinant nucleic acid molecule includes at
least one nucleic acid molecule of the present invention
operatively linked to one or more expression control sequences. As
used herein, the phrase "recombinant molecule" or "recombinant
nucleic acid molecule" primarily refers to a nucleic acid molecule
or nucleic acid sequence operatively linked to a expression control
sequence, but can be used interchangeably with the phrase "nucleic
acid molecule", when such nucleic acid molecule is a recombinant
molecule as discussed herein. According to the present invention,
the phrase "operatively linked" refers to linking a nucleic acid
molecule to an expression control sequence (e.g., a transcription
control sequence and/or a translation control sequence) in a manner
such that the molecule can be expressed when transfected (i.e.,
transformed, transduced, transfected, conjugated or conduced) into
a host cell. Transcription control sequences are sequences that
control the initiation, elongation, or termination of
transcription. Particularly important transcription control
sequences are those that control transcription initiation, such as
promoter, enhancer, operator and repressor sequences. Suitable
transcription control sequences include any transcription control
sequence that can function in a host cell or organism into which
the recombinant nucleic acid molecule is to be introduced.
[0399] Recombinant nucleic acid molecules of the present invention
can also contain additional regulatory sequences, such as
translation regulatory sequences, origins of replication, and other
regulatory sequences that are compatible with the recombinant cell.
In one embodiment, a recombinant molecule of the present invention,
including those that are integrated into the host cell chromosome,
also contains secretory signals (i.e., signal segment nucleic acid
sequences) to enable an expressed protein to be secreted from the
cell that produces the protein. Suitable signal segments include a
signal segment that is naturally associated with the protein to be
expressed or any heterologous signal segment capable of directing
the secretion of the protein according to the present invention. In
another embodiment, a recombinant molecule of the present invention
comprises a leader sequence to enable an expressed protein to be
delivered to and inserted into the membrane of a host cell.
Suitable leader sequences include a leader sequence that is
naturally associated with the protein, or any heterologous leader
sequence capable of directing the delivery and insertion of the
protein to the membrane of a cell.
[0400] One or more recombinant molecules of the present invention
can be used to produce an encoded product (e.g., an ACoAS) of the
present invention. In one embodiment, an encoded product is
produced by expressing a nucleic acid molecule as described herein
under conditions effective to produce the protein. A preferred
method to produce an encoded protein is by transfecting a host cell
with one or more recombinant molecules to form a recombinant cell.
Suitable host cells to transfect include, but are not limited to,
any bacterial, fungal (e.g., yeast), protist, microalgae, algae,
insect, plant or animal cell that can be transfected. In one
embodiment of the invention, a preferred host cell is a plant host
cell. Host cells can be either untransfected cells or cells that
are already transfected with at least one other recombinant nucleic
acid molecule.
[0401] According to the present invention, the term "transfection"
is used to refer to any method by which an exogenous nucleic acid
molecule (i.e., a recombinant nucleic acid molecule) can be
inserted into a cell. The term "transformation" can be used
interchangeably with the term "transfection" when such term is used
to refer to the introduction of nucleic acid molecules into
microbial cells, such as algae, bacteria and yeast, or into plant
cells. In microbial and plant systems, the term "transformation" is
used to describe an inherited change due to the acquisition of
exogenous nucleic acids by the microorganism or plant and is
essentially synonymous with the term "transfection." However, in
animal cells, transformation has acquired a second meaning which
can refer to changes in the growth properties of cells in culture
after they become cancerous, for example. Therefore, to avoid
confusion, the term "transfection" is preferably used with regard
to the introduction of exogenous nucleic acids into animal cells,
and the term "transfection" will be used herein to generally
encompass transfection of animal cells, and transformation of
microbial cells or plant cells, to the extent that the terms
pertain to the introduction of exogenous nucleic acids into a cell.
Therefore, transfection techniques include, but are not limited to,
transformation, particle bombardment, diffusion, active transport,
bath sonication, electroporation, microinjection, lipofection,
adsorption, infection and protoplast fusion.
[0402] It will be appreciated by one skilled in the art that use of
recombinant DNA technologies can improve control of expression of
transfected nucleic acid molecules by manipulating, for example,
the number of copies of the nucleic acid molecules within the host
cell, the efficiency with which those nucleic acid molecules are
transcribed, the efficiency with which the resultant transcripts
are translated, and the efficiency of post-translational
modifications. Additionally, the promoter sequence might be
genetically engineered to improve the level of expression as
compared to the native promoter. Recombinant techniques useful for
controlling the expression of nucleic acid molecules include, but
are not limited to, integration of the nucleic acid molecules into
one or more host cell chromosomes, addition of vector stability
sequences to plasmids, substitutions or modifications of
transcription control signals (e.g., promoters, operators,
enhancers), substitutions or modifications of translational control
signals (e.g., ribosome binding sites, Shine-Dalgarno sequences),
modification of nucleic acid molecules to correspond to the codon
usage of the host cell, and deletion of sequences that destabilize
transcripts.
[0403] Many genetic modifications useful for producing bioactive
molecules will be apparent to those of skill in the art, given the
present disclosure, and various other modifications have been
discussed previously herein. The present invention contemplates any
genetic modification related to a PUFA PKS system and/or accessory
protein as described herein which results in the production of a
desired bioactive molecule.
[0404] Bioactive molecules, according to the present invention,
include any molecules (compounds, products, etc.) that have a
biological activity, and that can be produced by a PUFA PKS system.
Such bioactive molecules can include, but are not limited to: a
polyunsaturated fatty acid (PUFA), an anti-inflammatory
formulation, a chemotherapeutic agent, an active excipient, an
osteoporosis drug, an anti-depressant, an anti-convulsant, an
anti-Heliobactor pylori drug, a drug for treatment of
neurodegenerative disease, a drug for treatment of degenerative
liver disease, an antibiotic, and a cholesterol lowering
formulation. One advantage of the PUFA PKS system of the present
invention is the ability of such a system to introduce
carbon-carbon double bonds in the cis configuration, and molecules
including a double bond at every third carbon. This ability can be
utilized to produce a variety of compounds.
[0405] Each publication, patent or patent application referenced
herein is incorporated herein by reference in its entirety.
[0406] The following examples are provided for the purpose of
illustration and are not intended to limit the scope of the present
invention.
EXAMPLES
[0407] General Introduction to Examples. Genes encoding PUFA
synthases have been identified in marine bacteria and in
thraustochytrid species. Several of these gene sets have been
expressed in E. coli and, when supplied with an appropriate PPTase,
the particular PUFA products of those enzymes can accumulate in
those cells. However, to the present inventors' knowledge, the
method of release of the PUFAs from these enzymes has not
previously been described. The release mechanism has implications
related to expression of PUFA synthase systems in heterologous host
organisms. It also can provide a direction to efforts aimed a
modulating the flux of carbon through that system and the eventual
amount of PUFAs that accumulate in heterologous, or native, host
organisms. Here the present inventors show that the products of the
Schizochytrium PUFA synthase (and, without being bound by theory,
likely all eukaryotic PUFA synthase systems, including all
thraustochytrid PUFA PKS systems) are free fatty acids, and that
the release of the free fatty acid is integral to the enzyme
complex itself. Further, in Schizochytrium, the PUFA FFA is
esterified to CoA prior to entry into the phospholipids (PL) and
triacylglycerols (TAG). The data described in the Examples below
indicate strategies for expression in heterologous host organisms
as well as for modification of PUFA accumulation in native host
organisms.
Example 1
[0408] This example describes the creation of a Schizochytrium FAS
knockout strain for biochemical studies.
[0409] Schizochytrium contains a single large gene that encodes the
FAS enzyme responsible for production of short chain saturated
fatty acids (described in U.S. Patent Application Publication No.
20050191679 A1). A Schizochytrium FAS knock out (FAS-KO) construct
was made using procedures described in U.S. Pat. No. 7,001,772. An
.about.10.0 kB EcoRV fragment of genomic DNA containing most of the
FAS Orf (from about 728 bp downstream of the presumed ATG start
codon to about 680 bp downstream of the stop codon) was cloned into
a Stratagene bluescript vector (pBSK) at the EcoRV site of the
multiple cloning region. An .about.3.5 kB internal BglII fragment
was removed from the cloned Schizochytrium DNA and replaced with an
.about.1.1 kB BamHI fragment from pTubZeo11-2 containing a Zeocin
resistance cassette (see U.S. Pat. No. 7,001,772, supra). The
plasmid (pJK878) was introduced into a cell wall defective strain
of Schizochytrium (denoted Ac66) via particle bombardment.
Transformants were initially selected by plating on media
containing Zeocin and supplemented with palmitic acid. A secondary
selection, failure to grow on plates not supplemented with palmitic
acid, was used to identify potential double crossover events in
which a portion of the FAS genomic region had been replaced by the
Zeocin resistant cassette. PCR and Southern blot analyses were used
to confirm that one of the transformants (labeled FAS-KO) had the
anticipated genomic structure. This strain was maintained by
growing in media supplemented with 500 uM palmitic acid. A similar
strategy, i.e. insertion of a Zeocin resistance cassette into one
of the genes encoding a subunit of the Schizochytrium PUFA
synthase, was employed to inactivate that enzyme in the
Schizochytrium Ac66 strain. In this case the medium is supplemented
with 500 uM DHA. Whole cells and cell free extracts of these
strains were used in subsequent biochemical studies (see Examples
below).
Example 2
[0410] The following example describes the general protocol for
preparation of cell free extracts of Schizochytrium Ac66, and PUFA
synthase KO and FAS-KO strains derived from Schizochytrium
Ac66.
[0411] An example of a protocol for preparation of cell free
homogenates (CFH) from the cell wall deficient strains of
Schizochytrium is as follows. Cells were grown in A50-3 medium and
then diluted into M2B medium. The media used for growing the KO
strains were supplemented with the appropriate fatty acid. Cells
were grown to an OD600 nm of >.about.2.5 and <.about.5 in the
M2B media. Cells in 50 mL of culture medium were collected by
centrifugation (table top centrifuge--.about.1200 rpm.times.4
minutes) in 50 mL plastic tubes. The supernatant was decanted and
the cells resuspended 5 mL Buffer A (100 mM Phosphate pH 7.2, 10%
(w/v) glycerol, 1 mM EDTA and 2 mM DTT) and centrifuged as before.
The supernatant was discarded and the cells resuspended in ice cold
5 mL Buffer A. The suspension was sonicated (Ultrasonic Processor
Model GE130 with microtip, Pulser at 2 seconds, .about.1 Watt power
setting) with tube on ice for 1.5 minutes. The sample was checked
by microscopy to ensure that all of the cells were broken. The CFH
was aliquoted in 200 uL portions into 0.5 mL PCR tubes with caps
and frozen by dropping into liquid N.sub.2. Samples were stored at
-74.degree. C. until needed.
Example 3
[0412] This example describes the general conditions for in vitro
FAS and PUFA synthase activity assays.
[0413] An example of a protocol for in vitro activity assays of
both FAS and PUFA synthase activities is as follows. In a final
volume of 100 uL, mix the enzyme preparation and Buffer A (volume
of these 2 components=90 uL) plus the following components added as
a cocktail (in 10 uL) to yield the final concentrations indicated
in parenthesis: malonyl-CoA (50 uM--a mixture of cold and
malonyl-2-.sup.14C-CoA such that the final concentration of
radiolabel is 0.65 .mu.Ci/mL), NADH (1 mM), NADPH (1 mM) and
acetyl-CoA (10 uM). These components and additional components can
be adjusted depending on the requirements of the particular
experiments. The assay reactions are carried out in glass tubes in
a room temperature (.about.21.degree. C.) water bath. The time of
incubation is dependant on the experimental requirements. The
reactions are stopped by one of two methods depending on the
work-up protocol. For conversion of fatty acids to fatty acid
methyl-esters (FAMEs) using an acidic method, the reaction is
stopped by adding the FAME reagent (see below). For extraction of
lipids without derivatization, the reaction is stopped by addition
of 125 uL of isopropanol:acetic acid (4:1 v/v) (see below).
[0414] Acidic FAME protocol: Stop the reaction by adding 2.0 mL of
4% HCl in methanol plus 50 uL toluene, seal the glass tubes with
Teflon lined caps and heat at 100.degree. C. for 1 hr. Cool to room
temperature, add 1.0 mL of hexane and 0.5 mL water, vortex then let
separate. If desired, remove a portion for liquid scintillation
counting (LSC). Transfer .about.600 uL of organic phase to a new
tube and remove the solvent under N.sub.2. Dissolve the residue in
50 uL hexane and spot onto either Silica gel 60 A TLC plates
(develop with hexane:diethyl-ether:acetic acid--70:30:2) or Silica
Gel G plates soaked in 10% AgNO.sub.3/90% acetonitrile (activated
for 30 min at 100.degree. C. prior to use) (develop
w/hexane:diethyl-ether/acetic acid --70:20:2). Let the plates air
dry and detect radioactive areas using phosphorimaging
technology.
[0415] HIP protocol--extraction of underivatized lipids: As
indicated above, stop the reaction by adding of 125 uL of
isopropanol:acetic acid (4:1 v/v) then add 2 mL of
hexane:isopropanol (3:2, v/v), vortex then add 1 mL of 6.7% (w/v)
sodium sulfate and vortex again. Let the phases separate. If
desired, remove a portion of the organic (upper) phase for LSC then
transfer the rest (.about.1.0 mL) to a new tube. Remove solvent
with N.sub.2 gas and dissolve the residue in 50 uL of hexane. Spot
the sample on a silica gel 60 A TLC plate and develop with
hexane:diethyl-ether:acetic acid (70:30:2). Let the plate air dry
and detect radioactive areas using phosphorimaging technology.
Example 4
[0416] The following example describes the results of in vitro
assays of FAS and PUFA synthase activities.
[0417] CFHs of Schizochytrium Ac66 and the PUFA synthase KO and
FAS-KO strains derived from Schizochytrium Ac66 were prepared and
assayed for FAS and PUFA synthase activities as described above
using the acidic FAME and silver TLC protocols. FIG. 1 shows the
results of those assays. The labeled bands on the image of the TLC
plate represent radioactivity incorporated into FAMEs (verified by
co-migration with standards as well as by HPLC separations). Lanes
1 and 2 show the profiles obtained using extracts from the Ac66
parental strain. Products of both the FAS (14:0 and 16:0 FAMEs) and
the PUFA synthase (DHA and DPA n-6) can be observed in these lanes.
The profiles obtained when the PUFA synthase enzyme has been
inactivated are shown in lanes 3 and 4. In this case, the DHA and
DPA n-6 FAMEs are not present. The profiles obtained when the FAS
is inactivated are shown in Lanes 5 and 6. In this case, the fatty
acids derived from the FAS, i.e. 14:0 and 16:0 and derivatives of
those fatty acids are missing. The data indicate that the FAS
activity has been severely, or completely, impaired in this FAS-KO
strain. The FAS-KO strain was used for further characterization of
the Schizochytrium PUFA synthesis and accumulation pathway.
Example 5
[0418] The following example describes additional characterization
of PUFA synthesis in Schizochytrium and provides evidence that the
initial product of the Schizochytrium PUFA synthase is a free fatty
acid (FFA).
[0419] Conversion of in vitro assay reaction products to FAMEs
using the acidic method is useful to determine incorporation of
radioactivity from malonyl-CoA into fatty acid moieties but it does
not show the molecular form of those fatty acids prior to that
derivatization. FIG. 2 shows the results of a time course of an in
vitro assay of the FAS-KO strain in which the lipids were extracted
using the HIP protocol described above (i.e., without conversion of
fatty acyl moieties to methyl esters) and then separated using
normal phase TLC. The positions on the plate where TAG and free
fatty acid (FFA) standards migrate are indicated to the left. In
this TLC system, FFA of different chain lengths and degrees of
unsaturation are not well separated. However, since the strain
utilized has little or no FAS activity FFAs in this zone are likely
to be derived from the PUFA synthase system. Additional evidence
supporting this is shown in FIG. 3. Here it is shown that
appearance of radiolabel in the FFA band during the in vitro assay
is dependant on the addition of NADPH. In contrast, NADH does not
support the reaction. This strict dependence on NADPH as a
reductant is also a characteristic of the PUFA synthase derived
from Shewanella SCRC2738 (FIG. 2C of Metz et al., Science
293:290-293 (2001)). In both FIGS. 2 and 3, a radiolabeled band
migrating slightly faster than the FFA band is apparent (labeled as
`Unknown`). Since the appearance of the band is independent of
addition of reductant (NADH or NADPH--see lane 5, FIG. 3), it is
unlikely to be associated with the PUFA synthase activity.
Additionally this band can be detected during a similar analysis of
strains in which the PUFA synthase has been inactivated (data not
shown). The data in FIGS. 2 and 3 suggest that the initial product
of the Schizochytrium PUFA synthase is a FFA. In FAS systems that
release their products as FFA (such as mammalian FAS), those FFA
are then esterified to CoA prior to entry into PL or TAG. The
activation of the FFA is carried out by acyl-CoA synthetases in a
reaction that requires ATP and Mg.sup.+2. The appearance of some
radioactivity in a TAG fraction late in a time course of the in
vitro reaction would be consistent with such a pathway in
Schizochytrium (due to residual ATP in the sample). This concept
was tested further (see below).
Example 6
[0420] The following example provides evidence in support of the
involvement of acyl-CoA synthetase reaction in the PUFA
accumulation pathway of Schizochytrium.
[0421] The effects of addition of ATP (2.5 mM) and Mg.sup.+2 (10
mM) on the in vitro assay products in samples from the
Schizochytrium FAS-KO are shown in FIG. 4. The samples were
incubated in the standard reaction mixture for 10 minutes and then
ATP and Mg.sup.+2 were added. The reactions were stopped at various
time points after the addition of ATP and Mg.sup.+2 (i.e., 0=no
addition, 10 and 30 sec, and 1, 3, 10 and 30 min). It can be seen
that radiolabel associated with the FFA band decreases and
radiolabel associated with the TAG band increases during the time
course. The radiolabel associated with the band labeled `Unknown`
is unaffected by the addition of ATP. These data are consistent
with the involvement of an ATP requiring reaction for migration of
labeled FFA into the TAG fraction.
[0422] Triacsin C has been characterized as a specific inhibitor of
acyl-CoA synthetases that activate long chain PUFAs (Knoll et al.,
1995). The effects of Triacsin C on the product profile during the
in vitro assays of FAS-KO samples were tested. The sample was
incubated in the standard cocktail containing various
concentrations of Triacsin C (0, 25, 100 or 200 uM) for 10 minutes
and then ATP and Mg.sup.+2 were added. The reaction was allowed to
proceed for an additional 20 minutes and then stopped and the
lipids extracted and separated by TLC using the HIP protocol. The
results are shown in FIG. 5. The addition of the Triacsin C at
higher concentrations blocked the loss of radiolabel from the FFA
band. These results are consistent with the involvement of an
acyl-CoA synthetase in the pathway.
Example 7
[0423] The following example describes in vitro assays of extracts
from E. coli expressing Schizochytrium Orf A, OrfBss (OrfB*), OrfC
and Nostoc HetI.
[0424] The data shown in the Examples above indicate that the PUFAs
in Schizochytrium are converted to the free fatty acid form prior
to entry into TAG and PL. Data indicating that the release of the
PUFA as a free fatty acid is an integral part of the PUFA synthase
enzyme is presented here. Schizochytrium native Orf A (nucleic acid
sequence represented by SEQ ID NO:1), OrfBss (also denoted OrfB*;
nucleic acid sequence represented by SEQ ID NO:37) and native OrfC
(nucleic acid sequence represented by SEQ ID NO:5) were cloned as
an artificial operon in a pET vector and expressed in E. coli as
described in U.S. Patent Application Publication No. 20050100995,
supra. Het I was cloned into a pACYC based vector and expressed in
those same cells. Cells were grown to an O.D. of .about.1 and IPTG
added (final concentration of 1 mM) to induce production of the T7
polymerase. Approximately 4 hours after induction, the cells were
harvested, washed with Buffer A and ruptured by two passages
through a French pressure cell. Aliquots of the homogenate were set
aside, and the rest centrifuged (5 k.times.g.times.5 min) to yield
Supernatant 1 (S1). Again, aliquots were set aside and the balance
of the material centrifuged at 100,000.times.g for 1 hour to yield
high speed pellet (P2) and high speed supernatant (S2) fractions.
The pellet fraction was resuspended in Buffer A to the volume
originally placed in the centrifuge tube. All of these fractions
were assayed using the general methods described above using the
acidic FAME/silver phase TLC workup or the HIP extraction of lipids
followed by separation on normal phase TLC. FIG. 6 shows the
results of those assays.
[0425] The acidic FAME analysis (FIG. 6A) shows that the primary
products of the in vitro assay are DHA and DPA n-6. The fraction
with the highest activity is the homogenate with much less activity
in the S1 and P2 fractions. Very little activity was detected in
the S2 fraction. It is of interest here that even in the CFH and S1
fractions, very little evidence of the products of the FAS system
can be detected (indicated by the arrow labeled as 16:0 in FIG.
6A). This is likely due to the high levels of expression of the
PUFA synthase enzyme components when using the T7 system. In
contrast, when similar assays were performed on extracts (CFH and
S1) from E. coli containing a cosmid encoding an EPA synthase from
Shewanella, the majority of the radioactivity on the TLC plate was
associated with FAS products (Metz et al., Science 293:290-293
(2001), FIG. 2B). Also, the endogenous E. coli FAS system is
composed of several individual soluble proteins and the FAS
activity remains in the supernatant fraction after high-speed
centrifugation (Metz et al., Science 293:290-293 (2001), FIG. 2B).
In contrast, the PUFA synthase activity shown in FIG. 6A partitions
into the pellet fraction after high-speed centrifugation.
[0426] The data in FIG. 6B show the results of assays of samples of
the same E. coli strain used for FIG. 6A, except that in the lipid
products were simply extracted with HIP (rather than being
converted to FAMES) prior to separation by TLC. Two fractions were
used, the CFH (left side of the figure) and the P2 (on the right
side). Amounts of the extracts used in the assays were adjusted so
that approximately equal amounts of radioactivity were incorporated
into lipids in the two cases. Also shown are the results in which
the reductant component (NADH and/or NADPH) of the assay cocktail
was varied as follows: Lane 1--only NADPH, Lane 2--only NADPH, Lane
3--both NADH and NADPH, and Lane 4--water was added instead of the
stock solutions containing either component. The data in FIG. 6B
show that most of the radiolabel that moves on the TLC plate
co-migrates with free fatty acid standards. Also, the appearance of
the major (FFA) band is dependant on the addition of NADPH to the
assay cocktail. The requirement for NADPH and the lack of
significant FAS activity in these fractions (especially the P2
fraction) indicate that the FFA is the product of the PUFA synthase
enzyme. Since only three genes from Schizochytrium (encoding Orfs
A, B and C) were expressed in this strain of E. coli (along with
Het I), the data indicate that release of the PUFA from the
synthase is an inherent property of that enzyme and not due to a
separate thioesterase enzyme.
[0427] A variety of data, important aspects of which have been
presented in the Examples above, indicate the following features of
PUFA synthesis and accumulation in Schizochytrium. The PUFA
synthase responsible for both DPAn-6 and DHA is encoded by Orfs A,
B and C as described in U.S. Pat. No. 6,566,583, Metz et al.,
Science 293:290-293 (2001), U.S. Patent Application Publication No.
20020194641, and PCT Publication No. WO 2006/135866. The ACP
domains of subunit A are activated by an endogenous PPTase. The
synthesis reaction uses malonyl-CoA as carbon source (acetyl-CoA
may or may not also be required) and NADPH as a reductant. The PUFA
products are released from the enzyme as FFAs and this release is
an inherent feature of the enzyme itself. The FFAs are esterified
to CoA in an ATP dependent reaction catalyzed by one or more
endogenous acyl-CoA synthetases. The PUFA-CoAs then serve as
substrates for the PL and TAG synthesis enzymes.
Example 8
[0428] The following example shows the expression of genes encoding
the Schizochytrium PUFA synthase (sOrf A, sOrfB and native OrfC,
see below) along with Het I in baker's yeast.
[0429] The Schizochytrium PUFA synthase genes and Het I were
expressed in yeast using materials obtained from Invitrogen. The
INVsc1 strain of Saccharomyces cerevisiae was used along with the
following transformation vectors: pYESLeu (sOrfA, SEQ ID NO:35,
encoding SEQ ID NO:2), pYES3/CT (sOrfB, SEQ ID NO:36, encoding SEQ
ID NO:4), pYES2/CT (OrfC, SEQ ID NO:5, encoding SEQ ID NO:6) and
pYESHis (HetI, SEQ ID NO:33, encoding SEQ ID NO:34). Some of the
vectors were modified to accommodate specific cloning requirements.
Appropriate selection media were used, depending on the particular
experiment. The genes were cloned, in each case, behind a GAL1
promoter and expression was induced by re-suspension of washed
cells in media containing galactose according to guidelines provide
by Invitrogen. Cells were grown at 30.degree. C. and harvested (by
centrifugation) at the indicated times after being transferred to
the induction medium. The cell pellets were freeze dried and FAMEs
were prepared using acidic methanol, extracted into hexane and
analyzed by GC.
[0430] Preliminary experiments indicated that expression of the
native form of OrfA (SEQ ID NO:1) and slightly modified native form
of OrfB (OrfB*, SEQ ID NO:37) in yeast did not result in production
of proteins of the expected size (correct mRNAs were also not
detected). In contrast, a protein of the expected size was detected
in cell in which the native form of OrfC (SEQ ID NO:5) was
expressed. The genes encoding OrfsA and B were resynthesized so
that their codon usage was more in line with those tolerated by
yeast (resynthesis was performed by Blue Heron, Inc.). These
synthetic genes are indicated herein as sOrfA (SEQ ID NO:35) and
sOrfB (SEQ ID NO:36). Expression of these genes in yeast resulted
in accumulation of proteins corresponding to the expected sizes of
Orf A and B, respectively.
[0431] FIG. 7 shows a comparison of the fatty acid profile from
yeast cells expressing the Schizochytrium PUFA synthase system
(sOrfA, sOrfB, OrfC and Het I) and one obtained from control cells
(lacking the sOrfA gene). Cells were collected .about.20 hrs after
induction. It can be seen that two novel FAME peaks have appeared
it the profile of the strain expressing the complete PUFA synthase
system. These two peaks were identified as DPA n-6 and DHA by
comparison of the elution time with authentic standards and
subsequently by MS analyses. As predicted from the inventors'
characterization of the Schizochytrium PUFA synthase, aside from
DHA and DPA n-6, no other novel peaks are evident in the
profile.
[0432] FIG. 8 shows the region of the GC chromatogram of FIG. 7,
which contains the PUFA FAMEs. Both the control cells and the cells
expressing the PUFA synthase contain a peak that elutes near the
DHA FAME. This has been identified as C26:0 FAME and (based on
literature references) is derived from sphingolipids. Although it
elutes close to the DHA peak, the resolution is sufficient so that
it does not interfere with the quantitation of DHA. The DPAn-6 peak
is well separated from other endogenous yeast lipids in the FAME
profile. In this particular example, the cells expressing the
Schizochytrium PUFA synthase system accumulated 2.4% DHA and 2.0%
DPAn-6 (as a percentage of the total FAMEs). The sum of DHA and DPA
n-6=4.4% of the measured fatty acids in the cells. The ratio of DHA
to DPA n-6 observed in the cells was .about.1.2:1.
[0433] The results presented above showing expression of the
Schizochytrium PUFA synthase in yeast provide a confirmation of the
pathway proposed in the previous applications as well as the
predictions in terms of the alterations to the fatty acid profiles
that can be expected in yeast and also in plants.
Example 9
[0434] The following example describes increasing the accumulation
of PUFAs in yeast expressing the Schizochytrium PUFA synthase by
co-expression of specific acyl-CoA synthetases.
[0435] The inventors have shown that in Schizochytrium, the FFA
products of its PUFA synthase are efficiently converted to acyl-CoA
by endogenous acyl-CoA synthetases (ACoASs) (see Examples above).
By examination of an EST database, the inventors identified 9
putative ACoASs that may be involved in conversion of the PUFAs to
the corresponding acyl-CoAs.
[0436] Briefly, the present inventors have examined a
Schizochytrium EST database consisting of sequences obtained from
.about.20,000 plasmids isolated from colonies picked from various
cDNA libraries for those ESTs that show homology to proteins with
known (or suspected) ACoAS activities. The inventors used the
VectorNTI program, Contig Express, to assemble these into contigs
(when two or more overlapping sequences were available) and edited
these based on the quality of the individual sequence information.
The results of this effort are summarized below. Eight different
contigs and one singlet (no overlapping sequences in the database)
were identified that were candidates for being associated with
ACoAS enzymes that can efficiently convert the product of the PUFA
synthase into the corresponding acyl-CoA. Using the EST data set as
a guide, the complete coding regions sequences for each candidate
was obtained and verified using various standard methods (e.g.,
sequencing of subclones of genomic DNA and PCR products derived
from genomic DNA).
Schizochytrium Acyl-CoA Synthetase (ACS) Coding Sequences and
Deduced Translations:
[0437] 1. Length=2004 nucleotides (not including the stop codon)
(SEQ ID NO:82). It is predicted to encode a 668 amino acid (SEQ ID
NO:83), 73.5 kDa, protein. The protein sequence has good homology
to known ACSs. The best Blast match is to a Thalassiosira
pseudonanna ACS (TplacA, Accession number: AAW58006) that has been
characterized and shown to have high activity with DHA (Tonon et
al., Plant Physiol. 2005 May; 138(1):402-8). The C-terminal three
amino acids of SEQ ID NO:83 are: SKL--a motif associated with
targeting of proteins to the peroxisome. This C-terminal motif is
also present in the Thalassiosira pseudonanna ACS mentioned
above.
[0438] 2. ScACS-2 (also denoted ScACoAS-2 or ACS-2): Length=2340
(not including the stop codon) nucleotides (SEQ ID NO:84). It is
predicted to encode a 780 amino acid (SEQ ID NO:85), 84.7 kDa,
protein. There is good homology over most of the putative protein
to known ACSs including the human examples, Lipidosin and Bubble
Gum.
[0439] 3. ScACS-3 (also denoted ScACoAS-3 or ACS-3): Length=2526
(not including the stop codon) nucleotides (SEQ ID NO:86). It is
predicted to encode an 842 amino acid (SEQ ID NO:87), 90.6 kDa,
protein. There is good homology over most of the putative protein
(particularly, the central .about.700 amino acids) with Bubble Gum
type ACS proteins.
[0440] 4. ScACS-4 (also denoted ScACoAS-4 or ACS-4): Length=2037
(not including the stop codon) nucleotides (SEQ ID NO:88). It is
predicted to encode a 679 amino acid (SEQ ID NO:89), 74.7 kDa
protein. There is good homology over most of the protein with known
ACS proteins, including examples from humans and other mammals.
[0441] 5. ScACS-5 (also denoted ScACoAS-5 or ACS-5): Length=1734
nucleotides (not including the stop codon) (SEQ ID NO:90). It is
predicted to encode a 578 amino acid (SEQ ID NO:91), 63.1 kDa,
protein. There is good homology over most of the protein with known
ACS proteins. The best Blast matches are to bacterial ACSs. The
C-terminal three amino acids of SEQ ID NO:91 are: SKL--a motif
associated with targeting of proteins to the peroxisome.
[0442] 6. ScACS-6 (also denoted ScACoAS-6 or ACS-6): Length=1806
(not including the stop codon) nucleotides (SEQ ID NO:92). It is
predicted to encode a 602 amino acid (SEQ ID NO:93), 66.0 kDa
protein. There is good homology over most of the protein with known
ACS proteins. The best Blast matches are to bacterial ACSs. The
C-terminal three amino acids of SEQ ID NO:93 are: SKL--a motif
associated with targeting of proteins to the peroxisome.
[0443] 7. ScACS-7 (also denoted ScACoAS-7 or ACS-7): Length=1920
(not including the stop codon) nucleotides (SEQ ID NO:94). It is
predicted to encode a 640 amino acid protein (SEQ ID NO:95), 70.4
kDa. There is good homology over most of the protein with known ACS
proteins. The best Blast matches are to bacterial ACSs.
[0444] 8. ScACS-8 (also denoted ScACoAS-8 or ACS-8): Length=1893
(not including the stop codon) nucleotides (SEQ ID NO:96). It is
predicted to encode a 631 amino acid (SEQ ID NO:97), 70.7 kDa
protein. The best Blast matches are to members of a fatty acid
transporter protein family that may also have ACoAS activity.
[0445] 9. ScACS-9 (also denoted ScACoAS-9 or ACS-9): Length=2950
(not including the stop codon) nucleotides (SEQ ID NO:98). It is
predicted to encode a 766 amino acid (SEQ ID NO:99), 84.1 kDa
protein. There is good homology over most of the protein with known
ACS proteins. The best Blast matches are to animal ACSs.
[0446] The inventors believed that enzymes present in heterologous
hosts of the PUFA synthases may not be able to efficiently process
the novel (for that organism) PUFA free fatty acids (FFAs), and
that co-expression of appropriate ACoAS(s) would result in
increased accumulation of the PUFAs in that host. Two of the
Schizochytrium candidate ACoASs described above (ScACS-1, SEQ ID
NO:82/83 and ScACS-2, SEQ ID NO:84/85) were individually expressed
in yeast that contained the genes encoding the Schizochytrium PUFA
synthase system (e.g., sOrfA, sOrfB and nOrfC, and HetI).
[0447] More specifically, the yeast expression system described in
the Examples above was modified to accommodate introduction of the
fifth ACoAS gene (i.e., the yeast also contained OrfsA, B and C of
the Schizochytrium PUFA synthase system and a PPTase (Het I from
Nostoc)) using 4 vectors. Yeast expression vectors in which two
genes can be cloned (the pESC vectors) were obtained from
Stratagene. These vectors are similar to and compatible with the
pYES vectors described above. Two genes, native OrfC (nOrfC, SEQ ID
NO:5) and HetI (SEQ ID NO:33), were cloned into one pESC vector,
while sOrfA (SEQ ID NO:35, sOrfB (SEQ ID NO:36) and the fifth gene
(ScACS-1 (SEQ ID NO:82) or ScACS-2 (SEQ ID NO:84)) were cloned into
pYES vectors. The four vectors were introduced into yeast and the
genes induced by resuspending cells in a galactose-containing
medium as describe above. Cells were grown at 30.degree. C. and
harvested 18 hours after induction. A summary of the FAME analysis
of these cells is shown in Table 1. The control cells contained all
4 vectors, but lacked the gene encoding Orf A. Co-expression of the
either one of the ScACOASs resulted in an increase in the
accumulation of DHA and DPA n-6 (approximately double the amount in
the control cells). This provides confirmation that the
accumulation of the products of the PUFA synthase in heterologous
host can be increased by co-expression of enzymes that may be more
efficient at utilization of those products. TABLE-US-00001 TABLE 1
30.degree. C., 18 hr Control ScACS-1 induction (PUFA genes) FAME
ScACS-2 Fatty Acid FAME (Area %) (Area %) FAME (Area %) C14:0* 1.7
1.8 2.0 C14.1 0.5 0.5 0.6 C15:0 0.5 0.5 0.5 C16:0* 17.1 16.5 15.5
C16:1* 40.7 38.8 38.5 C18:0* 4.7 4.3 4.2 C18:1 N9* 23.8 22.4 21.9
C18:1 N7 1.3 1.0 1.0 C24:0 0.1 0.1 0.1 C22:5 N6 1.3 2.5 3.1 C26:0
1.7 1.6 1.6 C22:6 N3* 2.0 3.8 3.9 DHA plus DPAn-6 3.3 6.3 7.0
[0448] In subsequent experiments, ScACS-3, ScACS-5, ScACS-6 and
ScACS-8 were also tested in yeast that contained the genes encoding
the Schizochytrium PUFA synthase system (e.g., sOrfA, sOrfB and
nOrfC, and HetI), using similar methods as described above.
Expression of each of ScACS-3, ScACS-5, or ScACS-8 all resulted in
increased DHA production in yeast as compared to in the absence of
the added acyl-CoA synthetase gene (data not shown).
[0449] As indicated above, the ScACS-8 shows homology to members of
a fatty acid transporter protein family that may also have ACS
activity. It is believed that these proteins are associated with
the plasma membrane and facilitate import of free fatty acids into
the cell and also convert them to the acyl-CoA derivatives. Enzymes
of this family may have particular utility when expressing PUFA
synthase systems, which release their products as free fatty acids,
in the plastids of plant cells. The outer envelope of the plastid
is thought to be derived from the plasma membrane and proteins
targeted to the plasma membrane (such as ScACS-8) may also be
targeted to the plastid outer envelope. If this is the case, these
fatty acid transport proteins (such as ScAC-8), may facilitate
export of the free fatty acid products of the PUFA synthase from
the plastid, and also convert them to the acyl-CoA derivatives. An
experiment to provide this acyl-CoA synthetase in plants that
express a Schizochytrium PUFA PKS system is described below.
Example 10
[0450] The following example demonstrates increasing levels of PUFA
in yeast expressing the Schizochytrium PUFA synthase, without or
with ScACoAS-1, by growth in the presence of cerulenin, which
inhibits the FAS pathway.
[0451] Both the PUFA synthase and FAS utilize malonyl-CoA as the
source of carbons for synthesis of their fatty acid products. In
addition, the acyl-CoA forms of fatty acids from both systems can
serve as substrates for enzymes which synthesize PL and TAG. As
discussed above, when both the PUFA synthase and FAS are present in
one organism, down regulation or inhibition of the FAS system is
expected to favor accumulation of PUFAs. Cerulenin is a
well-studied inhibitor of the condensation reactions of fatty acid
synthesis. Previous work indicated that PUFA synthases are
relatively less sensitive to inhibition by cerulenin than FAS
systems.
[0452] The present inventors tested the effects of cerulenin on
fatty acid profiles of the strains of yeast described in Example 8
as a model of the concept of reduction of FAS activity. The yeast
described in Example 9, which also contained an acyl
CoA-synthetase, were additionally tested in this system, to
determine whether the effects of the two strategies would
additively or synergistically increase PUFA production.
[0453] Initial experiments indicated the maximum effect (i.e., as
an increase in PUFAs as a percentage of the total fatty acid
profile) was obtained at a concentration of 4 uM cerulenin. The
cerulenin was added 4 hours after transfer to the galactose
induction medium. Cells were harvested 19 hr after transfer to
induction medium, freeze dried, FAMES prepared and analyzed by
GC.
[0454] The yeast strains tested were: [0455] Strain 5.5 contained
the PUFA synthase genes (sOrfA, sOrfB, OrfC and Het I), as
described in Example 8 above; and [0456] Strain 5.6 contained the
PUFA synthase gene set of Strain 5.5, plus the ScACoAS-1 (SEQ ID
NO:82), as described in Example 9 above.
[0457] Referring to Table 2, "0 Cer" indicates cerulenin was not
added, and "4 uM Cer" indicates the media was made to 4 uM
cerulenin 4 hours after transfer to the induction medium). Each
strain was evaluated for fatty acid production in the presence and
absence of the cerulenin, to evaluate the effect of the inhibition
of the FAS pathway on PUFA production. Table 2 shows the major
fatty acids detected in the GC profile (see also FIG. 11). The
values are given as a percentage of the total fatty acids detected.
DHA and DPAn-6, which are the products of the Schizochytrium PUFA
PKS system, were the only PUFAs present in the profiles. The sum of
DHA plus DPAn-6 is also indicated in Table 2. FIGS. 9 and 10
illustrate the amount of DHA (FIG. 9) or DHA and DPAn-6 (FIG. 10;
white bars are DHA; black bars are DHA+DPAn-6) produced by the
yeast, as a percentage of total FAME.
[0458] Yeast cells without the PUFA synthase genes do not make any
detectable PUFAs. Expression of the PUFA synthase system in yeast
in this experiment resulted in accumulation of 1.2% DHA. Inclusion
of the ScACoA-1 gene (SEQ ID NO:82) increased the DHA level to
4.1%. Growth of the cells with just the PUFA synthase system in the
presence of 4 uM cerulenin (inhibition of the FAS system) increased
the DHA level to 3.7%. When cells expressing both the PUFA synthase
and ScACoAS-1 genes were grown in 4 uM cerulenin (i.e., combined
expression of an acyl-CoA synthetase and inhibition of the FAS
system), the DHA level increased to 8.2% of total fatty acids. In
all of the samples, there was a corresponding increase in DPAn-6
accumulation. The sum of the DHA plus DPA n-6 in the samples is
also shown in Table 2 with the greatest amount (14.5% of total
fatty acids) present in Strain 5.6 grown in 4 uM cerulenin. It can
be seen that the effects of expressing the ACoA synthetase gene and
growth in the presence of cerulenin are additive. These data
support the invention proposed herein for increasing the
accumulation of PUFAs in heterologous hosts. TABLE-US-00002 TABLE 2
Strain 5.5 Strain 5.5 Strain 5.6 Strain 5.6 Fatty Acid 0 Cer 4 uM
Cer 0 Cer 4 uM Cer C14:0 1.5 0.0 1.7 0.0 C16:0 17.5 4.9 17.5 6.1
C16:1 43.4 38.4 41.7 34.8 C18:0 5.8 3.8 5.3 4.5 C18:1 N9 26.2 40.4
23.7 35.3 C18:1 N7 0.9 0.8 0.0 0.6 C22:5 N6 0.9 2.9 2.8 6.3 C26:0
2.0 2.9 1.9 2.4 C22:6 N3 1.3 3.7 4.1 8.2 DHA plus DPA N6 2.1 6.6
6.9 14.5
Example 11
[0459] The following example describes the identification of
additional accessory proteins or targets for use in increasing PUFA
production and/or accumulation in heterologous hosts.
[0460] Enzymes present in Schizochytrium efficiently utilize the
acyl-CoA forms of the products of the PUFA synthase to synthesize
phospholipid (PL) and triacylglycerol (TAG) molecules. However,
enzymes present in heterologous hosts may not carry out these
reactions with similar efficiency, since those PUFA-CoAs may not
typically be encountered by those organisms. For example,
expression of PL or TAG synthesis enzymes that efficiently
integrate the acyl-CoA products of the various PUFA synthases
(e.g., DHA-CoA, DPA n-6-CoA, EPA-CoA, or others) into PL or TAG
molecules in those heterologous hosts may result in the increased
ability to accumulate those products. In this regard,
Schizochytrium, or other organisms that produce PUFAs via the PUFA
synthase pathway, may serve as a good source of genes encoding
those enzymes. Accordingly, the present inventors propose the use
of several acyltransferase proteins that utilize PUFA-CoA as
substrates in forming PL or TAG (e.g., 3-glycerol-phosphate
acyltransferases (GPAT), lysophosphatidic acid acyltransferases
(LPAAT) and diacylglycerol acyltransferases (DAGAT)) or other
acyltransferases that may result in enrichment of PUFAs in PL or
TAG (e.g., phospholipid:diacylglycerol acyltransferases (PDAT)).
The identification of several such acyltransferases is described
below. A few of the candidates have been tested in yeast and are
tested in plants.
DAGAT Enzymes
[0461] The present inventors have examined the Schizochytrium EST
database for those ESTs that show homology to proteins with known
(or suspected) DAGAT activities. The inventors identified three
candidates as possible DAGAT enzymes for use in conjunction with a
PUFA PKS system, one of which is described below and has been shown
to be involved in the accumulation of free fatty acids into the TAG
molecules in Schizochytrium:
[0462] Schizochytrium DAGAT (also referred to as DAGAT-1 or
ScDAGAT-1)--Length of the coding region=1518 nucleotides (not
including the stop codon) (SEQ ID NO:100). It is predicted to
encode a 506 amino acid (SEQ ID NO:101), 57.4 kDa protein. There is
good homology over two thirds of the protein (starting at
.about.amino acid 170 and continuing to the C-terminus) with
proteins identified as DAGAT Type 2B enzymes. A Blast analysis of
the first one third of the protein sequence (amino acids 1 through
170) did not reveal significant homology to any proteins and did
not detect any Pfam matches.
[0463] Using the knock out technology described above in Example 1
for FAS in Schizochytrium, the inventors similarly knocked out the
DAGAT gene (comprising SEQ ID NO:100) in a Schizochytrium strain,
denoted B73-8. As shown in FIG. 13, inactivation of the DAGAT gene
in Schizochytrium significantly inhibited the accumulation of fatty
acids in the TAG. Specifically, inactivation of DAGAT resulted in
approximately an 80% reduction in mg FAME/gm biomass and
approximately a 90% reduction in TAG. Accordingly, the inventors
concluded that this DAGAT is the primary enzyme responsible for TAG
synthesis in Schizochytrium.
[0464] Accordingly, it is expected that expression of this nucleic
acid molecule in a host (e.g., yeast, plants) expressing a PUFA PKS
system described herein will increase the accumulation of free
fatty acids into the PL or TAG. A representative experiment
expressing this gene in a transgenic plant is described below.
LPAAT Enzymes
[0465] The present inventors have also examined the Schizochytrium
EST database for those ESTs that show homology to proteins with
known (or suspected) LPAAT activities. The inventors assembled
these into contigs (when two or more overlapping sequences were
available) and edited these based on the quality of the individual
sequence information as described above. The results of this effort
are summarized below. Three different contigs and one singlet (no
overlapping sequences in the database) were identified that were
particularly good candidates for being associated with LPAAT
enzymes. It is recognized that the enzymes encoded by these
sequences may have activities related to, but different from, the
putative LPAAT activity. In all four cases, a putative Orf
(including start and stop codons) were identified. It is recognized
that as more data are obtained that the precise sequence
representation, including identification of the endogenous start
codon, may change.
Schizochytrium LPAAT Candidates Identified by Analyses of EST
Database:
[0466] 1. ScLPAAT-1 Contig: Length=1478 nucleotides (SEQ ID
NO:102). It appears to include a full-length Orf of 927 nt
(including the stop codon, ScLPAAT-1 CDS, SEQ ID NO:103). A Blast
search using the translation of the CDS (SEQ ID NO:104) shows there
is good homology over most of the encoded protein to known and
putative acyltransferase proteins. The best matches are to proteins
from Arabidopsis. Pfam analysis indicates a large conserved central
domain related to the PlsC (1-acyl-sn-glycerol-3-phosphate
acyltransferase, i.e., LPAAT) family.
[0467] 2. ScLPAAT-2 Contig: Length=2112 nucleotides (SEQ ID
NO:105). It appears to include a full-length Orf of 1140 nt
(including the stop codon, ScLPAAT-2 CDS, SEQ ID NO:106). A Blast
search using the translation of the CDS (SEQ ID NO:107) shows there
is good homology over most of the encoded protein to known and
putative acyltransferase proteins. The best matches are to proteins
from Arabidopsis. Pfam analysis indicates a large conserved central
domain related to the PlsC (1-acyl-sn-glycerol-3-phosphate
acyltransferase, i.e., LPAAT) family.
[0468] 3. ScLPAAT-3 Contig: Length=1862 nucleotides (SEQ ID
NO:108). It appears to include a full-length Orf of 1323 nt
(including the stop codon, ScLPAAT-3 CDS, SEQ ID NO:109). A Blast
search using the translation of the CDS (SEQ ID NO:110) shows there
is good homology over the central part of the encoded protein to
known and putative acyltransferases. The best matches are to
proteins from mammals. Pfam analysis indicates a large conserved
central domain related to the PlsC (1-acyl-sn-glycerol-3-phosphate
acyltransferase, i.e., LPAAT) family.
[0469] 4. ScLPAAT-4 singlet: Length=794 nucleotides (SEQ ID
NO:111). It appears to include a full-length Orf of 756 nt
(including the stop codon, ScLPAAT-4 CDS, SEQ ID NO:112). A Blast
search using the translation of the CDS (SEQ ID NO:113) shows there
is good homology over much of the encoded protein to known and
putative acyltransferases. The best matches are to proteins from
birds and mammals. Pfam analysis indicates a large conserved
central domain related to the PlsC (1-acyl-sn-glycerol-3-phosphate
acyltransferase, i.e., LPAAT) family.
[0470] ScLPAAT-1 has been cloned expressed in yeast and plants.
Additional DAGAT or LPAAT Enzymes
[0471] The inventors have also examined the Crypthecodinium cohnii
EST database for those EST's that show homology to proteins with
known or suspected DAGAT or LPAAT activities. The results of this
effort are summarized below.
A) Crypthecodinium cohnii DAGAT Candidates Identified by Analyses
of EST Database:
[0472] 1. CA5_PTA.838.C: Length=817 nucleotides (SEQ ID NO:114).
There is good homology over the last 274 nucleotides of this
sequence to a Crypthecodinium acyltransferase sequence described in
PCT Publication No. WO 2004/087902.
[0473] 2. CA5_PTA.131.C1: Length=850 nucleotides (SEQ ID
NO:115).
[0474] 3. CA12_cot10.sub.--003a_h10: Length=663 nucleotides (SEQ ID
NO:116)
[0475] 4. CA12_cot10.sub.--001a_h02: Length=807 nucleotides (SEQ ID
NO:117)
[0476] 5. CA12_cot10.sub.--005b_g12: Length=765 nucleotides (SEQ ID
NO:118)
[0477] 6. CA12_cot50.sub.--005c_d07: Length=782 nucleotides (SEQ ID
NO:119)
B) Crypthecodinium cohnii LPAAT Candidates Identified by Analyses
of EST Database:
[0478] 1. CA12_cot10.sub.--003a_e11: Length=793 nucleotides (SEQ ID
NO:120)
[0479] 2. CA12_PTA.739.C1: Length=744 nucleotides (SEQ ID
NO:121)
[0480] Any one or more of the nucleic acid molecules described in
this Example can be used to transform any host cell, including to
produce any of the genetically modified organisms (e.g., plants or
microorganisms) described herein to further enhance PUFA
accumulation in an organism, and particularly, in an organism that
expresses a PUFA PKS system. These enzymes may also have utility
when expressed in a host organism that produces PUFAs by the
classical or standard fatty acid synthase pathway. Such constructs
can be used alone with the PUFA PKS system or in combination with
the other strategies for enhancing PUFA production and accumulation
in a host organism as described herein (e.g., with expression of an
acyl-CoA synthetase or with inhibition of the FAS pathway).
Additional acyltransferase sequences described in PCT Publication
No. WO 2004/087902 are also considered to be potentially useful in
the present invention and are incorporated herein by reference.
Example 12
[0481] The following example describes the expression of genes
encoding the Schizochytrium PUFA synthase (OrfA, OrfB* and OrfC)
along with Het I in Arabidopsis and the production of the target
PUFAs, DHA and DPAn-6, in the substantial absence of any detectable
intermediates or side products.
[0482] The Schizochytrium OrfA (nucleotide sequence represented by
SEQ ID NO:1), OrfB* (nucleotide sequence represented by SEQ ID
NO:37) and OrfC (nucleotide sequence represented by SEQ ID NO:5)
along with Het I (nucleotide sequence represented by SEQ ID NO:33)
were cloned (separately or in various combinations including all 4
genes on one superconstruct) into the appropriate binary vectors
for introduction of the genes into plants. Examples of such
constructs and vectors are described below (three expression
constructs) and also in Example 13 (one "superconstruct" for
4127).
Construction of 5720: Orf B* (Plastidic Expression)
[0483] The Orf B* (encoding SEQ ID NO:4), was restriction cloned
into an expression cassette under the control of the flax linin
promoter/terminator (U.S. Pat. No. 6,777,591). The linin promoter
controls the specific-temporal and tissue-specific expression of
the transgene(s) during seed development. Directly upstream and
in-frame of the Schizochytrium Orf B* was the plastid targeting
sequence derived from Brassica napus acyl-ACP thioesterase
(PT-signal peptide), to target Orf B* to the plastid The plant
binary vector also contained an existing E. coli phosphomannose
isomerase gene (Miles and Guest, 1984, Gene 32: 41-48) driven by
the ubiquitin promoter/terminator from Petroselinum crispum
(Kawalleck et al., 1993, Plant Mol. Bio., 21:673-684) between the
left and right border sequences for positive selection (Haldrup et
al., 1998, Plant Mol. Biol. 37:287-296).
Construction of 4107: HetI and Orf C (Plastidic Expression)
[0484] The Schizochytrium Orf C (nucleotide sequence represented by
SEQ ID NO:5) along with HetI (nucleotide sequence represented by
SEQ ID NO:33) were cloned into expression cassettes under the
control of a flax linin promoter/terminator (U.S. Pat. No.
6,777,591). The linin promoter controls the specific-temporal and
tissue-specific expression of the transgene(s) during seed
development. Directly upstream and in-frame of the Schizochytrium
Orf C and HetI was the plastid targeting sequence derived from
Brassica napus acyl-ACP thioesterase (PT-signal peptide), to target
the PUFA synthase and PPTase to the plastid. Both expression
cassettes were then assembled into one plant binary vector
containing a pat gene conferring host plant phosphinothricine
resistance (Wohlleben et al., 1988, Gene 70:25-37) driven by the
ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck
et al., 1993, Plant Mol. Bio., 21:673-684) between the left and
right border sequences.
Construction of 4757: Orf A (Plastidic Expression)
[0485] The Schizochytrium Orf A (nucleotide sequence represented by
SEQ ID NO:1) was cloned into expression cassettes under the control
of a flax linin promoter/terminator (U.S. Pat. No. 6,777,591). The
linin promoter controls the specific-temporal and tissue-specific
expression of the transgene(s) during seed development. Directly
upstream and in-frame of the Schizochytrium Orf A was the plastid
targeting sequence derived from Brassica napus acyl-ACP
thioesterase (PT-signal peptide), to target the PUFA synthase and
PPTase to the plastid. The expression cassette was contained within
a plant binary vector containing a nptII gene conferring host plant
kanamycin resistance driven by the MAS promoter/terminator between
the left and right border sequences.
[0486] In one example, transgenes were cloned into three separate
expression cassettes: a construct denoted 5720 (containing OrfB*,
encoding SEQ ID NO:4), a construct denoted 4107 (containing OrfC,
encoding SEQ ID NO:6 and HetI, encoding SEQ ID NO:34) and a
construct denoted 4757 (containing OrfA, containing SEQ ID NO:2),
as described above. In each construct, the gene was cloned. For
directing the proteins to the plastid, additional 5' sequences
encoding a plastid targeting sequence derived from a Brassica napus
acyl-ACP thioesterase were located directly upstream of Orfs A, B*,
C and HetI. The amino acid sequence of the encoded targeting
peptide is: MLKLSCNVTNHLHTFSFFSDSSLFIPVNRRTLAVS (SEQ ID NO:81). The
nucleotide sequences encoding this peptide were placed in-frame
with the start methionine codons of each PUFA synthase Orf, as well
as the engineered start codon (ATG) of Het I. In other constructs,
where localization of the PUFA synthase was targeted to the
cytoplasm of plant cells, no additional protein encoding sequences
were appended to the 5' end of the Orfs.
[0487] Standard methods were used for introduction of the genes
into Arabidopsis (floral dipping into suspension of Agrobacterium
strains containing the appropriate vectors, as described in Clough
et al., 1998, Plant J. 16: 735-743). The details of the methods are
described in Example 13 below. Seeds obtained from those plants
were plated on selective medium and allowed to germinate. Some of
the plants that grew were taken to maturity and the seeds analyzed
for PUFA content. Based on PUFA content some of those seeds were
taken forward to the next generation. Pooled seeds obtained from
those plants were analyzed for their fatty acid content. The target
PUFAs expected from these transgenic plants were docosahexaenoic
acid (DHA) and docosapentaenoic acid (DPAn-6), which are the
primary PUFAs produced by the Schizochytrium PUFA PKS system from
which the genes used to transform the plants were derived.
[0488] Results from one exemplary fatty acid analysis in one of the
exemplary transgenic plant lines is shown in FIG. 13. The top panel
of FIG. 13 shows the typical fatty acid profile of wild type
Arabidopsis seeds as represented by GC separation and FID detection
of FAMEs prepared from a pooled seed sample. The predominant fatty
acids are: 16:0, 18:0, 16:1, 18:1, 20:1, 20:2 and 22:1. No DHA or
DPA n-6 are present in the samples from wild type seed.
[0489] The lower panel of FIG. 13 shows the fatty acid profile of a
pooled seed sample from one of the exemplary transgenic Arabidopsis
lines (line 263) expressing the Schizochytrium PUFA synthase genes
and the Het I gene, introduced from three separate expression
cassettes (5720, 4107 and 4757) all targeted to the plastid, as
described above. Referring to the fatty acid profile of Line 263,
it is readily observed that two FAME peaks are present in the
profile from the transgenic plant seeds that are not present in the
profile from wild type seeds. The elution pattern of these two
peaks exactly corresponds to the elution of authentic DHA and
DPAn-6 (using FAMEs prepared from Schizochytrium oil as standards,
as well as a commercially purchased DHA standard from NuCheck
Prep). In this particular example, the DHA peak represents 0.8% of
total calculated FAMEs while the DPA n-6 peak represents 1.7%. The
sum of novel PUFAs is 2.5% of total FAMEs.
[0490] Experiments with other transgenic plant lines yielded
similar results. For example, another transgenic line, denoted 269,
which was transformed with the same constructs and in the same
manner as the 263 line, produced approximately 0.75% DHA or total
calculated FAMEs, and 1.41% DPAn-6 of total calculated FAMEs) (data
not shown).
[0491] Moreover, multiple other transgenic Arabidopsis plants
produced using the same nucleic acid molecules described above also
produced the target PUFAs, regardless of whether they were produced
using constructs providing the PUFA PKS genes and the HetI PPTase
on separate constructs, combination constructs, or a single
superconstruct (data shown below in Example 13).
[0492] In addition, transgenic plants targeting the PUFA PKS genes
to the cytosol all expressed the target PUFAs (data not shown in
detail). For example, a plant line expressing the Schizochytrium
PUFA PKS plus HetI in the cytosol introduced on three separate
expression cassettes as described above (without the plastid
targeting sequence) produced approximately 0.45% DHA and
approximately 0.8% DPA as a percentage of total FAME. In another
example, a plant line expressing the Schizochytrium PUFA PKS plus
HetI in the cytosol introduced on a single superconstruct (similar
to that described in Example 13 below) produced approximately
0.2-0.3% DHA and approximately 0.5% DPA as a percentage of total
FAME.
[0493] The appearance of DHA and DPAn-6 in the seed fatty acid
profile shown in FIG. 13 (and as observed in other transgenic
lines, some of which are described above) demonstrates that
introduced Schizochytrium PUFA synthase system functions when
expressed in the plant cell and that the proteins can be targeted
to the plastid. In addition, the inventors have confirmed that the
proteins can also be targeted to the cytosol, or both the plastid
and the cytosol, and produce PUFAs. As predicted from the
biochemical and heterologous expression data in other hosts (e.g.,
in E. coli and in yeast) the only novel fatty acids detected in the
profile of the seed from the transgenic plants are DHA and DPAn-6
(i.e., the fatty acid profile is substantially free of
contaminating intermediate or side products resulting from the PUFA
production enzyme system), further illustrating the advantages of
the PUFA PKS system over the standard pathway enzymes for the
production of PUFAs in a plant.
Examples 13(a)-13(j)
[0494] The following examples describe the use of various
strategies described herein (including combinations of strategies)
for increasing the production and/or accumulation of PUFAs in
plants.
[0495] Specifically, the following examples describe the expression
of genes encoding the Schizochytrium PUFA synthase (nOrfA, Orf B*
and nOrfC) along with Het I in Arabidopsis seeds, alone or in
combination with other accessory proteins and/or genetic
modification strategies to enhance PUFA production and
accumulation. Specifically, the Schizochytrium PUFA synthase and
Het I are expressed in plants alone or in combination with: (1) a
gene encoding an acyl-CoA synthetase (ACS), or (2) with genetic
elements intended to inhibit endogenous FAS activity. In addition,
an example of the combined use of the Schizochytrium PUFA synthase
and Het I in combination with expression of an ACS gene and a
genetic element intended to inhibit endogenous FAS activity is
shown. Finally, examples of expression of acyltransferases,
including DAGAT and/or LPAAT, alone or in combination with the
expression of one or more acyl-CoA synthetases and genetic elements
intended to inhibit endogenous FAS activity are described below.
The strategies outlined here illustrate the ability to enact of any
of the concepts described in the previous examples in plants.
Materials and Methods for Example 13(a)-(j)
(1) Constructs
Construction of Construct 4127: PT-Signal Peptide:nORFA, PT-Signal
Peptide:nORFB*, PT-Signal Peptide:HetI, Pt-SIGNAL PEPTIDE:nORFC
(Plastid Targeted Expression of Schizochytrium PUFA Synthase with
HetI)
[0496] The Schizochytrium native OrfA (nOrfA, represented by SEQ ID
NO:1, encoding SEQ ID NO:2), synthetic (resynthesized) OrfB*
(OrfB*, represented by SEQ ID NO:37 and encoding SEQ ID NO:4) and
native OrfC (nOrfC, represented by SEQ ID NO:5 and encoding SEQ ID
NO:6), along with HetI from Nostoc (represented by SEQ ID NO:33 and
encoding SEQ ID NO:34) were cloned into expression cassettes under
the control of a flax linin promoter/terminator (see U.S. Pat. No.
6,777,591 with regard to the promoter/terminator). The linin
promoter controls the specific-temporal and tissue-specific
expression of the transgene(s) during seed development. Directly
upstream and in-frame of the Schizochytrium Orfs A, B*, C and HetI
was the plastid targeting sequence derived from Brassica napus
acyl-ACP thioesterase (referred to herein as a PT-signal peptide,
the amino acid sequence of which is represented by SEQ ID NO:81),
also described in Example 12, to target the PUFA synthase and
PPTase to the plastid. All four expression cassettes were then
assembled into one plant binary vector containing a pat gene
conferring host plant phosphinothricine resistance (Wohlleben et
al., 1988, Gene 70:25-37) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant Mol. Bio., 21:673-684) between the left and right
border sequences.
Construction of 5723: ACS-1 (Cytosolic Expression)
[0497] For expression of an acyl-CoA synthetase, a separate plant
binary vector was constructed to express the nucleic acid sequence
for Schizochytrium ACS-1 (SEQ ID NO:82, encoding SEQ ID NO:83). The
ACS-1, with appropriate restriction sites engineered at the 5' and
3' ends was sub-cloned and sequenced. The ACS-1 was then
restriction cloned into an expression cassette under the control of
the flax linin promoter/terminator (U.S. Pat. No. 6,777,591) into a
plant binary vector containing the E. coli phosphomannose isomerase
gene (Miles and Guest, 1984, Gene 32:+41-48) driven by the
ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck
et al., 1993, Plant Mol. Bio., 21:673-684) between the left and
right border sequences for positive selection (Haldrup et al.,
1998, Plant Mol. Biol. 37:287-296).
[0498] Similar constructs were also produced for the expression of
the acyl-CoA synthetases referred to herein as ACS-2 (SEQ ID
NO:84/85) and ACS-8 (SEQ ID NO:96/97), 5724 and 5730 respectively.
In one aspect, the acyl-CoA synthetase sequences were combined with
nucleic acid molecules encoding a DAGAT (SEQ ID NO:100/101) and/or
LPAAT (SEQ ID NO:102/103/104), as described below.
Construction of 5727: KAS II RNAi with CHSA Intron (Cytosolic
Expression of KAS II RNAi with Intron)
[0499] For FAS inhibition, a separate plant binary vector was
constructed to attenuate the expression of KAS II. In this case, a
499 bp region of the nuclear encoded KAS II transcript encoded by
the At1g74960 locus (Carlsson et al., 2002, Plant J. 29: 761-770)
was targeted by RNA interference (RNAi) with an intervening intron
derived from the petunia chalcone synthase A (CHSA) gene (McGinnis
et al., 2005, Methods in Enzymology 392:1-24; Koes et al., 1989,
Gene 81: 245-257). The KAS II RNAi with CHSA intron (represented by
SEQ ID NO:122) was cloned into a plant binary vector between the
linin promoter/terminator (U.S. Pat. No. 6,777,591) in a plant
binary vector containing the E. coli phosphomannose isomerase gene
(Miles and Guest, 1984, Gene 32: 41-48) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant Mol. Bio., 21:673-684) between the left and right
border sequences for positive selection (Haldrup et al., 1998,
Plant Mol. Biol. 37:287-296).
Construction of 5729: KAS III Antisense RNA (Cytosolic Expression
of KAS III Antisense RNA)
[0500] For FAS inhibition, a separate plant binary vector was
constructed to attenuate the expression of KAS III. In this case, a
1210 bp antisense KAS III sequence derived from the nuclear encoded
transcript encoded by the At1g62640 locus (Yamada et al., 2002,
GenBank Accession AY091275) was targeted. The KAS III antisense
sequence (represented herein by SEQ ID NO:125) was cloned into a
plant binary vector between the linin promoter/terminator (U.S.
Pat. No. 6,777,591) in a plant binary vector containing the
phosphomannose isomerase gene (Miles and Guest, 1984, Gene 32:
41-48) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant Mol. Bio.,
21:673-684) between the left and right border sequences for
positive selection (Haldrup et al., 1998, Plant Mol. Biol.
37:287-296).
Construction of 5731: ACS-1 and KAS II RNAi with Intron (Cytosolic
Expression)
[0501] For expression of an acyl-CoA synthetase combined with FAS
inhibition, a separate plant binary vector was constructed to
attenuate the expression of KAS II and to express the nucleic acid
sequence for Schizochytrium ACS-1 (SEQ ID NO:82, encoding SEQ ID
NO:83). For this construct a double expression cassette of ACS-1
and KAS II RNAi with intron were expressed under the control of the
flax linin promoter/terminator (U.S. Pat. No. 6,777,591) into a
plant binary vector containing the E. coli phosphomannose isomerase
gene (Miles and Guest, 1984, Gene 32: 41-48) driven by the
ubiquitin promoter/terminator from Petroselinum crispum (Kawalleck
et al., 1993, Plant Mol. Bio., 21:673-684) between the left and
right border sequences for positive selection (Haldrup et al.,
1998, Plant Mol. Biol. 37:287-296).
Construction of 5732: ACS-1 and Antisense KAS II (Cytosolic
Expression)
[0502] For expression of an acyl-CoA synthetase combined with FAS
inhibition, a separate plant binary vector was constructed to
attenuate the expression of KAS II and to express the nucleic acid
sequence for Schizochytrium ACS-1 (SEQ ID NO:82, encoding SEQ ID
NO:83). For this construct a double expression cassette of ACS-1
and KAS II antisense with intron (KASII antisense sequence
represented herein by SEQ ID NO:123) were expressed under the
control of the flax linin promoter/terminator (U.S. Pat. No.
6,777,591) into a plant binary vector containing the E. coli
phosphomannose isomerase gene (Miles and Guest, 1984, Gene 32:
41-48) driven by the ubiquitin promoter/terminator from
Petroselinum crispum (Kawalleck et al., 1993, Plant Mol. Bio.,
21:673-684) between the left and right border sequences for
positive selection (Haldrup et al., 1998, Plant Mol. Biol.
37:287-296).
Construction of 5733: ACS-1 and KAS III RNAi (Cytosolic
Expression)
[0503] For expression of an acyl-CoA synthetase combined with FAS
inhibition, a separate plant binary vector was constructed to
attenuate the expression of KAS III and to express the nucleic acid
sequence for Schizochytrium ACS-1 (SEQ ID NO:82, encoding SEQ ID
NO:83). For this construct a double expression cassette of ACS-1
and KAS III RNAi (KASIII RNAi sequence represented herein by SEQ ID
NO:124) were expressed under the control of the flax linin
promoter/terminator (U.S. Pat. No. 6,777,591) into a plant binary
vector containing the E. coli phosphomannose isomerase gene (Miles
and Guest, 1984, Gene 32: 41-48) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant Mol. Bio., 21:673-684) between the left and right
border sequences for positive selection (Haldrup et al., 1998,
Plant Mol. Biol. 37:287-296).
Construction of 5734: ACS-1 and KAS III Antisense RNA (Cytosolic
Expression)
[0504] For expression of an acyl-CoA synthetase combined with FAS
inhibition, a separate plant binary vector was constructed to
attenuate the expression of KAS III and to express the nucleic acid
sequence for Schizochytrium ACS-1 (SEQ ID NO:82, encoding SEQ ID
NO:83). For this construct a double expression cassette of ACS-1
and KAS III antisense was expressed under the control of the flax
linin promoter/terminator (U.S. Pat. No. 6,777,591) into a plant
binary vector containing the E. coli phosphomannose isomerase gene
(Miles and Guest, 1984, Gene 32: 41-48) driven by the ubiquitin
promoter/terminator from Petroselinum crispum (Kawalleck et al.,
1993, Plant Mol. Bio., 21:673-684) between the left and right
border sequences for positive selection (Haldrup et al., 1998,
Plant Mol. Biol. 37:287-296).
Construction of 4793: DAGAT
[0505] For expression of a DAGAT, a separate plant binary vector
was constructed to express the nucleic acid sequence for
Schizochytrium DAGAT-1 (SEQ ID NO:100, encoding SEQ ID NO:101). The
Schizochytrium DAGAT (nucleotide sequence represented by SEQ ID
NO:100) was cloned into expression cassettes under the control of a
flax linin promoter/terminator (U.S. Pat. No. 6,777,591). The linin
promoter controls the specific-temporal and tissue-specific
expression of the transgene(s) during seed development. The
expression cassette was contained within a plant binary vector
containing a nptII gene conferring host plant kanamycin resistance
driven by the MAS promoter/terminator between the left and right
border sequences.
Construction of 4794: DAGAT and ACS-8
[0506] For expression of a DAGAT and an acyl-CoA synthetase, a
separate plant binary vector was constructed to express: (1) the
nucleic acid sequence for Schizochytrium DAGAT (SEQ ID NO:100,
encoding SEQ ID NO:101, and (2) the nucleic acid sequence for
Schizochytrium ACS-8 (SEQ ID NO:96, encoding SEQ ID NO:97). For
this construct a double expression cassette of ACS-8 and DAGAT was
expressed under the control of the flax linin promoter/terminator
(U.S. Pat. No. 6,777,591) into a plant binary vector containing a
nptII gene conferring host plant kanamycin resistance driven by the
MAS promoter/terminator between the left and right border
sequences.
Construction of 4795: LPAAT and DAGAT
[0507] For expression of an LPAAT and a DAGAT, a separate plant
binary vector was constructed to express: (1) the nucleic acid
sequence for Schizochytrium LPAAT (SEQ ID NO:103, encoding SEQ ID
NO:104, and (2) the nucleic acid sequence for Schizochytrium
DAGAT-1 (SEQ ID NO:100, encoding SEQ ID NO:101). For this construct
a double expression cassette of LPAAT and DAGAT was expressed under
the control of the flax linin promoter/terminator (U.S. Pat. No.
6,777,591) into a plant binary vector containing a nptII gene
conferring host plant kanamycin resistance driven by the MAS
promoter/terminator between the left and right border
sequences.
Construction of 4796: ACS-8, LPAAT, and DAGAT
[0508] For expression of an acyl-CoA synthetase, LPAAT and DAGAT, a
separate plant binary vector was constructed to express: (1) the
nucleic acid sequence for Schizochytrium LPAAT (SEQ ID NO:103,
encoding SEQ ID NO:8104, (2) the nucleic acid sequence for
Schizochytrium DAGAT-1 (SEQ ID NO:100, encoding SEQ ID NO:101), and
(3) the nucleic acid sequence for Schizochytrium ACS-8 (SEQ ID
NO:96, encoding SEQ ID NO:97). For this construct a triple
expression cassette of ACS-8, LPAAT and DAGAT was expressed under
the control of the flax linin promoter/terminator (U.S. Pat. No.
6,777,591) into a plant binary vector containing a nptII gene
conferring host plant kanamycin resistance driven by the MAS
promoter/terminator between the left and right border
sequences.
(2) Transformation of Arabidopsis
[0509] The integrity of all plant binary vectors were confirmed by
diagnostic restriction digests and sequence analysis. Isolated
plasmids were then used to transform competent Agrobacterium strain
EH101 (Hood et al., 1986, J. Bacteriol. 144: 732-743) by
electroporation (25 .mu.F, 2.5 kV, 200.OMEGA.). Recombinant
Agrobacterium were plated on AB-spectinomycin/kanamycin
(20.times.AB salts, 2 M glucose, 0.25 mg/ml FeSo.sub.4.7H.sub.2O, 1
M MgSo.sub.4, 1 M CaCl.sub.2) and a single colony was used to
inoculate 5 ml of AB-spectinomycin/kanamycin broth. These cultures
were grown overnight at 28.degree. C. The recombinant Agrobacteria
containing the 4127 plasmid were then used to transform wild type
C24 Arabidopsis thaliana plants by the flower dipping method
(Clough et al., 1998, Plant J. 16: 735-743). Seeds obtained from
these plants were plated on selective medium in the presence of
phosphinothricine and allowed to germinate. Positively identified
seedlings were transferred to soil and taken to maturity, after
which the seeds were analyzed for PUFA content.
[0510] For recombinant Agrobacterium containing the other plasmids
(5723, 5724, 5730, 5727, 5729, 5731, 5732, 5733, 5734, 4793, 4794,
4795, and/or 4796), transgenic 4127-Line 150 Arabidopsis thaliana
plants were re-transformed by the flower dipping method (Clough et
al., 1998, Plant J. 16: 735-743). Seeds obtained from these plants
were plated on selective medium in the presence of
phosphinothricine and mannose for double selection or
phosphinothricine, mannose and kanamycin or phosphinothricine and
kanamycin for triple selection, where appropriate, and allowed to
germinate. Positively identified seedlings were transferred to soil
and taken to maturity, after which the seeds were analyzed for PUFA
content.
Example 13a
[0511] This example describes production of DHA and DPAn-6 in
transgenic Arabidopsis thaliana seed expressing the Schizochytrium
PUFA synthase (OrfA, OrfB* and OrfC) along with Het I on a
superconstruct (4127).
[0512] GC-FAME analyses of pooled seeds from Arabidopsis plants
expressing the Schizochytrium PUFA synthase (OrfA, OrfB* and OrfC)
along with Het I (construct 4127) revealed significant levels of
the target PUFAs, DHAn-3 and DPAn-6, in their fatty acid content.
As shown in Table 3, one line in particular (4127-Line 150)
exhibited 0.6% DHAn-3 and 0.7% DPAn-6 for a combined 1.3%
Schizochytrium-type PUFA content. As expected, the control seeds
from the wild type (C24) background do not contain any detectable
levels of either DHAn-3 or DPAn-6. Subsequent expression analysis
of 4127-Line 150 performed by SDS-PAGE and Western blotting
revealed the recombinant seed expressed OrfA, OrfB*, OrfC and Het I
correctly targeted to the plastid (data not shown). Furthermore,
this phenotype was stable from analysis of the T2 generation
through until the analysis of the T4 generation, which served as a
positive control for determining if DHA and Schizochytrium PUFA
levels when various strategies described herein (including
combinations of strategies) were evaluated for increasing the
production and/or accumulation of PUFAs in plants. TABLE-US-00003
TABLE 3 DHA and DPA levels in mature wild type Arabidopsis seed in
comparison to transgenic seed expressing the Schizochytrium PUFA
synthase along with Hetl (plastid targeted) in T2 and T4 pooled
seeds populations selected from phosphinothricine positive plants.
The % DHAn-3 and % DPAn-6 were determined following GC separation
and FID detection of total calculated FAMEs. Phenotype % DHA % DPA
(C22:6 (C22:5 % DHA + Strategy Genotype Line Generation n-3) n-6)
DPA Negative Wild Type C24 N/A 0 0 0 control (pooled seed) ecotype
PUFA OrfA, OrfB*, 4127-Line T2 0.6 0.7 1.3 Synthase + Hetl OrfC,
Hetl 150 T4 0.6 0.6 1.2 (pooled seed)
Example 13b
[0513] This example describes the production of DHAn-3 and DPAn-6
in transgenic Arabidopsis thaliana seed expressing the
Schizochytrium PUFA synthase (Orf A, Orf B* and Orf C) with Het I
(4127) in combination with the Schizochytrium ScACS-1 gene (5723)
or ScACS-2 gene (5724).
[0514] Plants derived from 4127-Line 150 (see Example 13a) were
used for the introduction of the ScACS-1 construct (5723) or
ScACS-2 construct (5724) by Agrobacterium-mediated transformation
as described above. Following the selection of recombinant plants
in the presence of both phosphinothricine and mannose, seeds were
harvested and analyzed for fatty acid profiles by GC separation and
FID detection of FAMES prepared from pooled seed.
[0515] As an example, one line in particular expressing the
Schizochytrium PKS along with HetI in combination with ACS-1
(4127/5723-Line 514) exhibited 1.5% DHA and 0.9% DPAn-6 for a
combined 2.4% Schizochytrium PUFA content in the total fatty acid
profile (Table 4). This represented a 2.5 fold increase in DHAn-3
content over the 4127-Line 150 positive control. Similar results
were observed in a line which expressed the Schizochytrium PKS
along with HetI in combination with ACS-2 (4127/5724-Line 552)
which exhibited a 1.8 fold increase in DHA n-3 content in
comparison to the positive control. Furthermore, a shift in the DHA
to DPA ratio from approximately 0.85:1.0 in the T2 generation or
1.0:1.0 in the T4 generation of 4127-Line 150 to 1.7:1.0 in the
ACS-1 and 1.2:1.0 in the ACS-2 lines was observed. In all
transgenic seed analyzed, the only novel fatty acids detected in
the profile were DHA n-3 or DPA n-6. TABLE-US-00004 TABLE 4 DHAn-3
and DPAn-6 levels in mature wild type and transgenic Arabidopsis
seed expressing the Schizochytrium PUFA synthase along with Hetl
(plastid targeted) in comparison to transgenic seed combining
Schizochytrium PUFA synthase along with Hetl (plastid targeted)
expression and with expression of Schizochytrium ACS-1 or ACS-2, in
pooled seeds. The % DHA n-3 and % DPA n-6 were determined following
GC separation and FID detection of total calculated FAMEs.
Phenotype % DHA % DPA (C22:6 (C22:5 % DHA + Strategy Genotype Line
Generation n-3) n-6) DPA Negative Wild Type C24 N/A 0 0 0 control
(pooled seed) ecotype Positive OrfA, OrfB*, 4127-Line T2 0.6 0.7
1.3 Control OrfC, Hetl 150 T4 0.6 0.6 1.2 (pooled seed) AcylCoAS
OrfA, OrfB*, 4127/5723- T4/T2 1.5 0.9 2.4 Expression OrfC, Hetl,
Line 514 ACS-1 (pooled seed) OrfA, OrfB*, 4127/5724- 1.1 0.9 2.0
OrfC, Hetl, Line 552 ACS-2 (pooled seed)
Example 13c
[0516] This example describes the production of DHA and DPAn-6 in
transgenic Arabidopsis thaliana seed expressing the Schizochytrium
PUFA synthase (OrfA, OrfB* and OrfC) along with Het I, combined
with FAS inhibition through the attenuation of KAS II using RNA
interference (RNAi).
[0517] Plants derived from 4127-Line 150 were used for the
introduction of the KAS II RNAi with intron (construct 5727) by
Agrobacterium-mediated transformation as described above. Following
the selection of recombinant plants in the presence of both
phosphinothricine and mannose, seeds were harvested and analyzed
for fatty acid profiles by GC separation and FID detection of FAMES
prepared from pooled seed.
[0518] As an example, one line in particular (4127/5727-Line 1097)
exhibited 1.3% DHA n-3 and 1.2% DPA n-6 for a combined 2.5%
Schizochytrium PUFA content in the total fatty acid profile (Table
5). This represented more than a 2.1 fold increase in DHA content
over the 4127-Line 150 positive control. Subsequently, single-seeds
from 4127/5727-Line 1097 were individually analyzed by GC
separation and FID detection of total calculated FAMEs.
[0519] Following this analysis it was observed that seed within
this population exhibited up to 2.0% DHA n-3 and 1.6% DPAn-6 for a
combined 3.6% Schizochytrium PUFA content in the fatty acid profile
(Table 5). This represents a 3.3 fold increase in DHA content and a
3-fold increase in Schizochytrium PUFA content over the 4127-Line
150 positive control. Furthermore, a shift in the DHA to DPA ratio
from 0.85:1.0 in the T2 generation or 1.0:1.0 in the T4 generation
of 4127-Line 150 to 1.25:1.0 or greater in the FAS inhibition line
was observed. The single seed average was consistent with the
pooled sample with respect to % DHA n-3, % DPA n-6 and total %
(DHA+DPA) and differences within this population can be attributed
to segregation of the recombinant 4127 and 5727 loci in
co-transformed seed. In all transgenic seed analyzed, the only
novel fatty acids detected in the profile were DHA n-3 or DPA n-6.
TABLE-US-00005 TABLE 5 DHA and DPA levels in mature wild type and
transgenic Arabidopsis seed expressing the Schizochytrium PUFA
synthase along with Hetl (plastid targeted) in comparison to
transgenic seed combining Schizochytrium PUFA synthase along with
Hetl (plastid targeted) expression with KAS II attenuation in
pooled and single seeds. The % DHA n-3 and % DPA n-6 were
determined following GC separation and FID detection of total
calculated FAMEs. Phenotype % DHA % DPA (C22:6 (C22:5 % DHA +
Strategy Genotype Line Generation n-3) n-6) DPA Negative Wild Type
C24 N/A 0 0 0 control (pooled seed) ecotype Positive OrfA, OrfB*,
4127-Line T2 0.6 0.7 1.3 Control OrfC, Hetl 150 T4 0.6 0.6 1.2
(pooled seed) FAS OrfA, OrfB*, 4127/5727- T4/T2 1.3 1.2 2.5
inhibition OrfC, Hetl, Line 1097 KAS II RNAi with intron (pooled
seed) OrfA, OrfB*, 1097-7 0.7 0.7 1.4 OrfC, Hetl, 1097-9 0.7 0.8
1.5 KAS II RNAi 1097-2 0.9 0.9 1.8 with intron 1097-5 1.0 0.9 1.9
(single seed) 1097-6 1.0 1.1 2.1 1097-1 1.2 1.3 2.5 1097-8 1.3 1.3
2.6 1097-4 1.4 0.8 2.2 1097-10 1.4 1.2 2.6 1097-3 2.0 1.6 3.6
Single T4/T2 1.2 1.0 2.2 seed average
Example 13d
[0520] This example describes the production of DHA and DPAn-6 in
transgenic Arabidopsis thaliana seed expressing the Schizochytrium
PUFA synthase (OrfA, OrfB* and OrfC) along with Het I, combined
with FAS inhibition through the attenuation of KAS III using
antisense RNA.
[0521] Plants derived from 4127-Line 150 were used for the
introduction of the KAS III antisense construct (5129) by
Agrobacterium-mediated transformation as described above. Following
the selection of recombinant plants in the presence of both
phosphinothricine and mannose, seeds were harvested and analyzed
for fatty acid profiles by GC separation and FID detection of FAMES
prepared from pooled seed.
[0522] As an example, one line in particular (4127/5729-Line 1087)
exhibited 1.7% DHA n-3 and 1.2% DPA n-6 for a combined 2.9%
Schizochytrium PUFA content in the total fatty acid profile (Table
6). This represented a 2.8 fold increase in DHA content over the
4127-Line 150 positive control.
[0523] Subsequently, single-seeds from 4127/5729-Line 1087 were
individually analyzed by GC separation and FID detection of total
calculated FAMEs. Following this analysis it was observed that seed
within this population exhibited up to 2.4% DHA n-3 and 1.8% DPA
n-6 for a combined 4.2% Schizochytrium PUFA content in the fatty
acid profile (Table 6). This would represent a 4 fold increase in
DHA content and 3.2 fold increase in Schizochytrium PUFA content
over the 4127-Line 150 positive control. Furthermore, a shift in
the DHA to DPA ratio from 0.85:1.0 in the T2 generation or 1.0:1.0
in the T4 generation of 4127-Line 150 to 1.33:1.0 or greater in the
FAS inhibition line was observed. The single seed average was
consistent with the pooled sample with respect to % DHA n-3, % DPA
n-6 and total % (DHA+DPA) and differences within this population
can be attributed to segregation of the recombinant 4127 and 5729
loci in co-transformed seed. In all transgenic seed analyzed the
only novel fatty acids detected in the profile were DHA n-3 or DPA
n-6 as predicted from the previous biochemical and heterologous
expression data observed in E. coli and yeast. The GC-FAME
chromatogram obtained for analysis of the seed sample 1087-7 is
shown for reference in FIG. 14. TABLE-US-00006 TABLE 6 DHA and DPA
levels in mature wild type and transgenic Arabidopsis seed
expressing the Schizochytrium PUFA synthase along with Hetl
(plastid targeted) in comparison to transgenic seed combining
Schizochytrium PUFA synthase along with Hetl (plastid targeted)
expression with KAS III attenuation in pooled and single seeds. The
% DHA n-3 and % DPA n-6 were determined following GC separation and
FID detection of total calculated FAMEs. Phenotype % DHA % DPA
(C22:6 (C22:5 % DHA + Strategy Genotype Line Generation n-3) n-6)
DPA Negative Wild Type C24 N/A 0 0 0 control (pooled seed) ecotype
Positive OrfA, OrfB*, 4127-Line T2 0.6 0.7 1.3 Control OrfC, Hetl
150 T4 0.6 0.6 1.2 (pooled seed) FAS OrfA, OrfB*, 4127/5729- T4/T2
1.7 1.2 2.9 inhibition OrfC, Hetl, Line 1087 KAS III antisense RNA
(pooled seed) OrfA, OrfB*, 1087-9 0.9 1.0 1.9 OrfC, Hetl, 1087-4
1.0 1.1 2.1 KAS III 1087-2 1.1 0.9 2.0 antisense 1087-6 1.2 0.6 1.8
RNA 1087-1 1.3 1.1 2.4 (single seed) 1087-8 1.4 1.5 2.9 1087-3 1.7
1.1 2.8 1087-10 1.8 1.6 3.4 1087-5 2.0 1.6 3.6 1087-7 2.4 1.8 4.2
Single T4/T2 1.5 1.2 2.7 seed average
Example 13e
[0524] This example describes the production of DHA and DPAn-6 in
transgenic Arabidopsis thaliana seed expressing the Schizochytrium
PUFA synthase (OrfA, OrfB* and OrfC) along with Het I, combined
with both expression of the ScACS-1 gene and FAS inhibition through
the attenuation of KAS III using antisense RNA.
[0525] Plants derived from 4127-Line 150 were used for the
introduction of the ScACS-1 plus KAS II RNAi using construct 5731
by Agrobacterium-mediated transformation as described above.
Following the selection of recombinant plants in the presence of
both phosphinothricine and mannose, seeds were harvested and
analyzed for fatty acid profiles by GC separation and FID detection
of FAMES prepared from pooled seed.
[0526] As an example, one line (4127/5731-Line 1366) exhibited 1.9%
DHA and 1.9% DPA n-6 for a combined 3.8% Schizochytrium PUFA
content in the total fatty acid profile (Table 7). This represented
a 3.2 fold increase over the 4127-Line 150 positive control, a 1.3
fold increase over the ACS-1 strategy alone as observed in
4127/5723-Line 514 and a 1.5 fold increase compared to the KAS II
RNAi attenuation strategy alone as observed in 4127/5727-Line 1097
when comparing DHA content from pooled seed populations as
described in examples 13b and 13c (Tables 4 and 5),
respectively.
[0527] One would expect higher levels of DHA content to be observed
in single seeds within this population as a reflection of
segregation of the 4127 and 5731 loci amongst the pooled seed. In
all transgenic seed analyzed the only novel fatty acids detected in
the profile were DHA n-3 or DPA n-6 as predicted from the previous
biochemical and heterologous expression data observed in E. coli
and yeast. The GC-FAME chromatogram obtained for analysis of the
pooled seed sample 4127/5731-Line 1366 is shown for reference in
FIG. 15. TABLE-US-00007 TABLE 7 DHAn-3 and DPAn-6 levels in mature
wild type and transgenic Arabidopsis seed expressing the
Schizochytrium PUFA synthase along with Hetl (plastid targeted) in
comparison to transgenic seed combining Schizochytrium PUFA
synthase along with Hetl (plastid targeted) combined with
expression of Schizochytrium ACS-1 and FAS inhibition, in pooled
seeds. The % DHA n-3 and % DPA n-6 were determined following GC
separation and FID detection of total calculated FAMEs. Phenotype %
DHA % DPA (C22:6 (C22:5 % DHA + Strategy Genotype Line Generation
n-3) n-6) DPA Negative Wild Type C24 N/A 0 0 0 control (pooled
seed) ecotype Positive OrfA, OrfB*, 4127-Line T2 0.6 0.7 1.3
Control OrfC, Hetl 150 T4 0.6 0.6 1.2 (pooled seed) AcylCoAS OrfA,
OrfB*, 4127/5731- T4/T2 1.9 1.9 3.8 Expression OrfC, Hetl, Line and
FAS ACS-1, KAS 1366 inhibition II RNAi (pooled seed)
Example 13f
[0528] This example describes the production of DHA and DPAn-6 in
transgenic Arabidopsis thaliana seed expressing the Schizochytrium
PUFA synthase (OrfA, OrfB* and OrfC) along with Het I, combined
with expression of the Schizochytrium LPAAT.
[0529] Plants derived from 4127-Line 150 were used for the
introduction of the LPAAT construct (5725) by
Agrobacterium-mediated transformation as described above. Following
the selection of recombinant plants in the presence of both
phosphinothricine and mannose, seeds will be harvested and analyzed
for fatty acid profiles by GC separation and FID detection of FAMES
prepared from pooled seed.
[0530] It is expected that seeds from these plants will produce the
target PUFAs (DHA and DPAn-6). It is also expected that the levels
of DHA and/or DPAn-6 production will be increased as compared to
the PUFA PKS-expressing plant in the absence of the added LPAAT
construct.
Example 132
[0531] This example describes the production of DHA and DPAn-6 in
transgenic Arabidopsis thaliana seed expressing the Schizochytrium
PUFA synthase (OrfA, OrfB* and OrfC) along with Het I, combined
with expression of the Schizochytrium DAGAT and ACS-1, and FAS
inhibition through the attenuation of KAS II using RNAi or the
attenuation of KASIII using antisense.
[0532] Plants derived from 5731 (combined expression of ACS-1 and
FAS inhibition by KASII RNAi) were used for the introduction of the
DAGAT construct (4793) by Agrobacterium-mediated transformation as
described above. Similar plants were also produced on the 5734
background (combined expression of ACS-1 and FAS inhibition by
KASIII antisense). Following the selection of recombinant plants in
the presence of both phosphinothricine and mannose, seeds will be
harvested and analyzed for fatty acid profiles by GC separation and
FID detection of FAMES prepared from pooled seed.
[0533] It is expected that seeds from these plants will produce the
target PUFAs (DHA and DPAn-6). It is also expected that the levels
of DHA and/or DPAn-6 production will be increased as compared to
the PUFA PKS-expressing plant in the absence of the added DAGAT
construct and FAS inhibition.
Example 13h
[0534] This example describes the production of DHA and DPAn-6 in
transgenic Arabidopsis thaliana seed expressing the Schizochytrium
PUFA synthase (OrfA, OrfB* and OrfC) along with Het I, combined
with expression of the Schizochytrium DAGAT and ACS-8, further
combined with expression of the Schizochytrium ACS-1 and FAS
inhibition through the attenuation of KAS II using RNAi or the
attenuation of KASIII using antisense.
[0535] Plants derived from 5731 (combined expression of ACS-1 and
FAS inhibition by KASII RNAi) were used for the introduction of the
DAGAT/ACS-8 construct (4794) by Agrobacterium-mediated
transformation as described above. Similar plants were also
produced on the 5734 background (combined expression of ACS-1 and
FAS inhibition by KASIII antisense). Following the selection of
recombinant plants in the presence of both phosphinothricine and
mannose, seeds will be harvested and analyzed for fatty acid
profiles by GC separation and FID detection of FAMES prepared from
pooled seed.
[0536] It is expected that seeds from these plants will produce the
target PUFAs (DHA and DPAn-6). It is also expected that the levels
of DHA and/or DPAn-6 production will be increased as compared to
the PUFA PKS-expressing plant in the absence of the added
DAGAT/ACS-8 construct, the ACS-1 construct, and FAS inhibition.
Example 13i
[0537] This example describes the production of DHA and DPAn-6 in
transgenic Arabidopsis thaliana seed expressing the Schizochytrium
PUFA synthase (OrfA, OrfB* and OrfC) along with Het I, combined
with expression of the Schizochytrium LPAAT and Schizochytrium
DAGAT, further combined with expression of the Schizochytrium ACS-1
and FAS inhibition through the attenuation of KAS II using RNAi or
the attenuation of KASIII using antisense.
[0538] Plants derived from 5731 (combined expression of ACS-1 and
FAS inhibition by KASII RNAi) were used for the introduction of the
DAGAT/LPAAT construct (4795) by Agrobacterium-mediated
transformation as described above. Similar plants were also
produced on the 5734 background (combined expression of ACS-1 and
FAS inhibition by KASIII antisense). Following the selection of
recombinant plants in the presence of both phosphinothricine and
mannose, seeds will be harvested and analyzed for fatty acid
profiles by GC separation and FID detection of FAMES prepared from
pooled seed.
[0539] It is expected that seeds from these plants will produce the
target PUFAs (DHA and DPAn-6). It is also expected that the levels
of DHA and/or DPAn-6 production will be increased as compared to
the PUFA PKS-expressing plant in the absence of the added
DAGAT/LPAAT construct, the ACS-1 construct, and FAS inhibition.
Example 13j
[0540] This example describes the production of DHA and DPAn-6 in
transgenic Arabidopsis thaliana seed expressing the Schizochytrium
PUFA synthase (OrfA, OrfB* and OrfC) along with Het I, combined
with expression of the Schizochytrium LPAAT, Schizochytrium DAGAT,
and Schizochytrium ACS-8, further combined with expression of the
Schizochytrium ACS-1 and FAS inhibition through the attenuation of
KAS II using RNAi or the attenuation of KASIII using antisense.
[0541] Plants derived from 5731 (combined expression of ACS-1 and
FAS inhibition by KASII RNAi) were used for the introduction of the
DAGAT/LPAAT/ACS-8 construct (4796) by Agrobacterium-mediated
transformation as described above. Similar plants were also
produced on the 5734 background (combined expression of ACS-1 and
FAS inhibition by KASIII antisense). Following the selection of
recombinant plants in the presence of both phosphinothricine and
mannose, seeds will be harvested and analyzed for fatty acid
profiles by GC separation and FID detection of FAMES prepared from
pooled seed.
[0542] It is expected that seeds from these plants will produce the
target PUFAs (DHA and DPAn-6). It is also expected that the levels
of DHA and/or DPAn-6 production will be increased as compared to
the PUFA PKS-expressing plant in the absence of the added
DAGAT/LPAAT/ACS-8 construct, the ACS-1 construct, and FAS
inhibition.
[0543] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070245431A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070245431A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References