`US 9,458,436 B2
`(10) Patent No.:
`(45) Date of Patent:
`*Oct. 4, 2016
`Cirpus et al.
`
`US009458436B2
`
`METHOD FOR PRODUCING
`POLYUNSATURATED FATTY ACIDS IN
`TRANSGENIC PLANTS
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`(54)
`
`(75)
`
`Inventors: Petra Cirpus, Mannheim (DE); Jfirg
`Bauer, Ludwigshafen (DE); Xiao Qiu,
`Saskatoon (CA); Guohai Wu,
`Saskatoon (CA); Nagamani Datla,
`Saskatoon (CA)
`
`(73)
`
`Assignee: BASF Plant Science GmbH,
`Ludwigshafen (DE)
`
`(*)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 1278 days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21)
`
`Appl. No.:
`
`10/590,457
`
`(22)
`
`PCT Filed:
`
`Feb. 23, 2005
`
`5,614,393 A
`6,043,411 A
`6,459,018 B1
`6,884,921 B2
`7,777,098 B2
`2004/0049805 A1
`2004/0053379 A1
`2004/0111763 A1
`2004/0172682 A1 *
`2008/0155705 A1
`2009/0222951 A1
`2010/0021976 A1
`
`3/1997 Thomas et 31.
`3/2000 Nishizawa et al.
`10/2002 Knutzon
`4/2005 Browse et 31.
`8/2010 Cirpus et al.
`3/2004 Lerchl et al.
`3/2004 Lerchl et al.
`6/2004 Heinz et al.
`9/2004 Kinney et al.
`6/2008 Zank et al.
`9/2009 Cirpus et al.
`1/2010 Lerchl et al.
`
`................ 800/281
`
`CA
`DE
`DE
`EP
`EP
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`FOREIGN PATENT DOCUMENTS
`
`2 485 060
`101 02 337 A1
`102 19 203
`0 550 162
`0 794 250
`WO-91/13972
`WO-93/06712
`WO-93/11245
`WO-94/11516
`WO-94/18337
`WO-95/18222 A1
`WO-96/21022
`
`11/2003
`7/2002
`11/2003
`7/1993
`9/1997
`9/1991
`4/1993
`6/1993
`5/1994
`8/1994
`7/1995
`7/1996
`
`(Continued)
`
`OTHER PUBLICATIONS
`
`PCT No.:
`
`PCT/EP2005/001863
`
`§ 371 (C)(1)s
`(2), (4) Date:
`
`Aug. 25, 2006
`
`PCT Pub. No.: W02005/083093
`
`PCT Pub. Date: Sep. 9, 2005
`
`(86)
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`(87)
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`(65)
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`(30)
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`Prior Publication Data
`
`GeneSeq Accession ABV74261, Lerchl et a1 (Mar. 28, 2003).*
`
`US 2009/0222951 A1
`
`Sep. 3, 2009
`
`Foreign Application Priority Data
`
`(Continued)
`
`Feb. 27, 2004
`Mar. 13, 2004
`Apr. 8, 2004
`May 14, 2004
`Jul. 16, 2004
`Dec. 24, 2004
`
`(DE) ........................ 10 2004 009 457
`(DE) ........... 10 2004 012 370
`
`(DE) ........... 10 2004 017 518
`
`(DE) .................. 10 2004 024 014
`
`(EP) ............. PCT/EP2004/007957
`
`
`(DE) .................. 10 2004 062 543
`
`(51)
`
`(52)
`
`(58)
`
`Int. Cl.
`C12N 15/82
`C12N 9/02
`A23D 9/00
`A61K 8/92
`
`A61Q 19/00
`C12N 9/10
`A61K 8/36
`A61K 31/202
`U.S. Cl.
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`CPC ............... C12N 9/0071 (2013.01), A23D 9/00
`(2013.01), A61K 8/361 (2013.01), A61K 8/922
`(2013.01), A61K 31/202 (2013.01), A61Q
`19/00 (2013.01), C12N 9/0083 (2013.01),
`C12N 9/1029 (2013.01), C12N 15/8247
`(2013.01), A61K 2800/86 (2013.01), C12Y
`114/19 (2013.01)
`
`Field of Classification Search
`None
`
`Primary Examiner 7 Elizabeth McElwain
`
`(74) Attorney, Agent, or Firm 7 Drinker Biddle & Reath
`LLP
`
`(57)
`
`ABSTRACT
`
`The present invention relates to a process for the production
`of polyunsaturated fatty acids in the seed of transgenic
`plants by introducing, into the organism, nucleic acids which
`encode polypeptides with a (03-desaturase, A12-desaturase,
`A6-desaturase, A6-elongase, A5-desaturase, A5-elongase
`and/or A4-desaturase activity. The invention furthermore
`relates to recombinant nucleic acid molecules comprising
`the nucleic acid sequences which encode the aforementioned
`polypeptides, either jointly or individually, and transgenic
`plants which comprise the aforementioned recombinant
`nucleic acid molecules. Furthermore, the invention relates to
`the generation of a transgenic plant and to oils, lipids and/or
`fatty acids with an elevated content of polyunsaturated fatty
`acids, in particular arachidonic acid, eicosapentaenoic acid
`and/or docosahexaenoic acid, as the result of the expression
`of the elongases and desaturases used in the process accord-
`ing to the invention.
`
`See application file for complete search history.
`
`25 Claims, 33 Drawing Sheets
`
`1 of 290
`1 of 290
`
`CSIRO Exhibit 1013
`CSIRO Exhibit 1013
`
`
`
`US 9,458,436 B2
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`Page 2
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`(56)
`
`References Cited
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`FOREIGN PATENT DOCUMENTS
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`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
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`WO-97/21340
`WO-97/30582
`WO-98/46763
`WO-98/46764
`WO-98/46765
`WO-98/46776
`WO-99/27111
`WO-99/64616
`WO-00/12720
`WO-00/21557
`WO-01/59128
`WO-02/08401
`WO-02/44320
`WO-02/057464 A2
`WO-02/077213
`WO-02/081668 A2
`WO-02/092540 A1
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`U.S. Patent
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`Oct. 4, 2016
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`Sheet 2 of 33
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`US 9,458,436 B2
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`Figure 2:
`
`Substrate specificity of the A5-elongase (SEQ ID NO: 53) with regard to
`
`different fatty acids
`
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`Sheet 3 of 33
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`US 9,458,436 B2
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`Figure 3:
`
`Reconstitution of DHA biosynthesis in yeast starting from 20:5033.
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`Sheet 4 of 33
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`US 9,458,436 B2
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`Figure 4:
`
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`Oct. 4, 2016
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`Sheet 5 of 33
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`US 9,458,436 B2
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`Figure 5:
`
`Fatty acid composition (in mol%) of transgenic yeasts which had been
`
`transformed with the vectors pYesB-OmELO3/pYesZ-EgD4 or pYesB—
`
`OmELOB/pYesZ-EgD4+pESCLeu-PtD5. The yeast cells were cultured in
`
`minimal medium without tryptophan and uracil/ and leucin in the presence
`of 250 pM 20:5A5"‘-“"“'17 and 18:4“3-‘2-‘5, respectively. The fatty acid methyl
`
`esters were obtained from cell sediments by acid methanolysis and ana-
`
`lyzed via GLC. Each value represents the mean (n=4) i standard deviation.
`
`pYes3-OmELO/pYesZ-EgD4
`
`pYesS-OmELO/pYesZ-EgD4 EgD4
`
`+ pESCLeu-PtDS
`
`Fatty acids
`
`Feeding of 20:5A5’3’H'M'17
`
`Feeding of 1824‘36'9'12'15
`
`16:0
`
`16:1 A9
`
`18:0
`
`18:1Ag
`
`18:1 A“
`
`9.35 :l: 1.61
`
`14.70 i 2.72
`
`5.11 i 1.09
`
`1949:301
`
`18.93 i 2.71
`
`1194“,”;15
`
`-
`
`20:1 A“
`
`20:1 4“
`
`3.24 i 0.41
`
`11.13: 2.07
`
`20.442.11.14.”
`
`-
`
`20.545.411.14,"
`
`6.913: 1.10
`
`22:4‘“°’“""”
`
`-
`
`22:5 “mm”
`
`8.77 :t 132
`
`22:6“""“"’3""’9
`
`2.73 :l: 0.39
`
`7.35 i 1.37
`
`10.02 3: 1.81
`
`4.27: 1.21
`
`10.81 i 1.95
`
`11.61 i 1.48
`
`7.79 i 1.29
`
`1.56 i 0.23
`
`4.40 i 0.78
`
`30.05 :I: 3.16
`
`3.72 :l: 0.59
`
`5.71 i 1.30
`
`1.10 :l: 0.27
`
`0.58 i 0.10
`
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`
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`U.S. Patent
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`Oct. 4, 2016
`
`Sheet 6 of 33
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`US 9,458,436 B2
`
`
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`9 of 290
`9 of 290
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`U.S. Patent
`
`Oct. 4, 2016
`
`Sheet 7 of 33
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`US 9,458,436 B2
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`U.S. Patent
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`Oct. 4, 2016
`
`Sheet 8 of 33
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`US 9,458,436 B2
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`Oct. 4, 2016
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`Sheet 9 of 33
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`US 9,458,436 B2
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`12 of 290
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`13 of 290
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`U.S. Patent
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`Oct. 4, 2016
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`Sheet 11 of 33
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`US 9,458,436 B2
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`
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`2mm:nemaw.09.58.
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`14 of 290
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`
`
`
`
`
`U.S. Patent
`
`Oct. 4, 2016
`
`Sheet 12 of 33
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`US 9,458,436 B2
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`Figure 12: Desaturation of y-linolenic acid (18:2 (DB-fatty acid) to give a-linolenic acid
`
`(18:3 m3-fatty acid) by Pi-omegaBDes.
`
`mOMEGA3+1&2
`
`
`
`15 of 290
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`U.S. Patent
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`Oct. 4, 2016
`
`Sheet 13 of 33
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`US 9,458,436 B2
`
`Figure 13: Desaturation of y—Iinolenic acid (18:2 (DB-fatty acid) to give stearidonic acid
`
`(18:4 cos-fatty acid) by Pi-omega3Des.
`
`piOMEGA3 + y—18:3
`
`
`
`16 of 290
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`
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`U.S. Patent
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`Oct. 4, 2016
`
`Sheet 14 of 33
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`US 9,458,436 B2
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`Figure 14: Desaturation of 020:2 coS-fatty acid to give 020:3 m3-fatty acid by Pi-
`
`omega3Des.
`
`piOMEGA3 + c20:2’“”4
`
`17 of 290
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`U.S. Patent
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`Oct. 4, 2016
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`Sheet 15 of 33
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`US 9,458,436 B2
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`Figure 15: Desaturation of 020:3 cos-fatty acid to give 020:4 m3-fatty acid by Pi-
`
`omega3Des.
`
`piOMEGA3 + 020:3“.11.“
`
`
`
`18 of 290
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`
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`U.S. Patent
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`Oct. 4, 2016
`
`Sheet 16 of 33
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`US 9,458,436 B2
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`Figure 16: Desaturation of arachidonic acid (020:4 cue-fatty acid) to give eicosapen-
`
`taenoic acid (C20:5 m3-fatty acid) by Pi-omegaBDes.
`
`piOMEGA3 + 020:4A5,8,11,14
`
`
`
`19 of 290
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`Oct. 4, 2016
`
`Sheet 17 of 33
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`US 9,458,436 B2
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`Figure 17: Desaturation of docosatetraenoic acid (022:4 (DB-fatty acid) to give
`
`docosapentaenoic acid (022:5 coB-fatty acid) by Pi-omegaBDes.
`
`piOMEGA3 + 22:4
`
`
`
`20 of 290
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`U S. Patent
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`Oct. 4, 2016
`
`Sheet 18 of 33
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`US 9,458,436 B2
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`‘12
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`21 of 290
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`
`U.S. Patent
`
`Oct. 4, 2016
`
`Sheet 19 of 33
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`US 9,458,436 B2
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`Figure19:Desaturationofphospholipid-boundarachidonicacidtogiveEPAbyPi-Omega3Des
`
`
`
`Fraction
`
`00000
`[\COLOVCO
`
`pammesap 17:02 %
`
`
`
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`: E O E
`
`
`
`22 of 290
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`Oct. 4, 2016
`
`Sheet 20 of 33
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`US 9,458,436 B2
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`Figure 20: Conversion of linoleic acid (arrow) to give y-linolenic acid (7-1823) by Ot-
`De56.1.
`
`Absorption mAU
`
`16:016:1
`
`Retention time
`
`23 of 290
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`
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`U.S. Patent
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`Oct. 4, 2016
`
`Sheet 21 of 33
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`US 9,458,436 B2
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`Figure 21: Conversion of linoleic acid and a-linolenic acid (A and C), and reconstitution
`
`of the ARA and EPA synthetic pathways, respectively, in yeast (B and D) in
`
`the presence of OtDG.1.
`
`A) OtDB+LA
`
`GLA
`
`LA
`
`B) OtDG+PSE1+PtD5+LA
`
`
`
`C) OtD6+ALA
`
`D) OtDB+PSE1+PtD5+ALA
`
`
`
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`U.S. Patent
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`Oct. 4, 2016
`
`Sheet 22 of 33
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`US 9,458,436 B2
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`Figure 22: Expression of ELO(X|) in yeast
`
`Absorption in mA
`
`A) ELO (XI) without fatty acid feeding
`
`
`
`B) ELO (XI) + 18:4A6,9,12,15 (250 ,uM)
`
`
` Retention time in min
`
`C) ELO (XI) + 20:5 (500 ,uM)
`
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`Oct. 4, 2016
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`Sheet 23 of 33
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`US 9,458,436 B2
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`Figure 23:
`
`Absorption in mA
`
`A) EIO (Ci) without fatty acid feeding
`
`B) ELO (Ci) + 18:4 (250 ,uM)
`
`
`
` Retention time in minutes
`
`C) ELO (Ci) + 20:5 (500 ,uM)
`
`26 of 290
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`Oct. 4, 2016
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`Sheet 24 of 33
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`US 9,458,436 B2
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`Figure 24: Elongation of eicosapentaenolc acid by OtEIo1 (B) and OtElo1.2 (D), re-
`
`spectively. The controls (A, C) do not show the elongation product (22:5033).
`
`20:5A5,8_11,14.17
`
`
`
`22;5A7,10,13,16,19 H
`20:5A5.8,11,14,17
`
`25
`
`21.5
`
`30
`
`325
`
`35
`
`375
`
`40
`
`42.5
`
`45
`
`HDSmd
`
`18:1A9
`
`27.5
`
`3D
`
`325
`
`35
`
`37,5
`
`40
`
`425
`
`45
`
`Retention time (min)
`
`20:5(03
`
`pYESZ
`
`
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`Sheet 25 of 33
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`US 9,458,436 B2
`
`Figure 25: Elongation of arachidonic acid by OtElo1 (B) and OtE|o1.2 (D), respectively.
`
`The controls (A, C) do not show the elongation product (22:4m6).
`
`FIDSigwal
`
`
`
`20:445.5.11,“
`
`
`
`22;4A7.1o,13,16
`_
`
`20:445.5.11.14
`
`Retention time (min)
`
`
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`28 of 290
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`Oct. 4, 2016
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`Sheet 26 of 33
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`US 9,458,436 B2
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`Figure 26: Elongation of 20:5n-3 by the elongases At3g06470.
`
`Absorption in mA
`
`20:5(n-3)
`
`22:5(n-3)
`
` 5
`
`13
`
`15
`
`17
`
`19
`
`7
`
`9
`
`11
`
`Retention time in minutes
`
`29 of 290
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`Oct. 4, 2016
`
`Sheet 27 0f 33
`
`US 9,458,436 B2
`
`Figure 27:
`
`Substrate specificity of the Xenopus Elongase (A), Ciona Elongase (B) und
`
`Oncorhynchus Elongase (C)
`
`A)
`
`Wv
`
`C)
`
`20
`
`15
`
`10
`
`25
`
`20
`
`15
`
`10
`
`60
`
`45
`
`30
`
`15
`
`100
`
`75
`
`50
`
`25
`
`100
`
`75
`
`50
`
`25
`
`100
`
`75
`
`50
`
`25
`
`
`
`
`
`absoluteelongation(%)
`
`
`
`
`
`relativeelongation(%)
`
`l
`
`A9
`
`ll
`
`A6
`
`’LQBJLALU‘
`
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`
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`
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`
`Fatty acid
`
`30 of 290
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`Oct. 4, 2016
`
`Sheet 28 of 33
`
`US 9,458,436 B2
`
`Figure 28:
`
`Substrate specificity of the Ostreococcus A5-elongase (A), the Ostreococ-
`
`cus A6—elongase (B), the Thalassiosira A5-elongase (C) and the Thalas-
`
`siosira Ostreococcus AB-elongase (D)
`
`A)
`
`B)
`
`9
`
`D)
`
`
`
`
`
`absoluteelongation(%)
`
`_
`Fatty Acu d
`
`so
`
`45
`
`3o
`
`15
`
`0
`
`25
`
`20
`
`15
`
`10
`
`5
`
`O
`
`1 5
`
`10
`
`5
`
`0
`80
`
`60
`
`40
`
`20
`
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`203w6
`1&3w6
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`183w3
`
`204w6l>20:5w3a
`
`182w6l>
`
`100
`
`75
`
`50
`
`25
`
`100
`
`75
`
`50
`
`25
`
`100
`
`75
`
`50
`
`25
`
`100
`
`75
`
`5o
`
`25
`
`226w3
`
`
`
`
`
`relativeelongation(%)
`
`31 of 290
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`Oct. 4, 2016
`
`Sheet 29 of 33
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`US 9,458,436 B2
`
`Figure 29:
`
`Expression of the Phaeodactyium tricornutum A6—elongase (PtELOG) in
`yeast. A) shows the elongation of the 018:3“5'9'12 fatty acid and B) the
`elongation of the C18:3“"'9'12'15 fatty acid
`
`A)
`
`
`
`3)
`
`18:1A9
`18:4A6'9'121'15
`
`
`32 of 290
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`Oct. 4, 2016
`
`Sheet 30 of 33
`
`US 9,458,436 B2
`
`Figure 30:
`
`Figure 30 shows the substrate specificity of PtEL06 with regard to the sub-
`strates fed.
`
`PtELO6 specificity
`
`100
`
`-----------------------------------------------------------------------------------------------------
`
`80
`
`60
`
`40
`
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`US 9,458,436 B2
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`US 9,458,436 B2
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`1
`METHOD FOR PRODUCING
`POLYUNSATURATED FATTY ACIDS IN
`TRANSGENIC PLANTS
`
`RELATED APPLICATIONS
`
`This application is a national stage application (under 35
`U.S.C. 371) ofPCT/EP2005/001863 filed Feb. 23, 2005, and
`claims benefit of German application 10 2004 009 457.8
`filed Feb. 27, 2004; German application 10 2004 012 370.5
`filed Mar. 13, 2004; German application 10 2004 017 518.7
`filed Apr. 8, 2004; German application 10 2004 024 014.0
`filed May 14, 2004; PCT application PCT/EP2004/07957
`filed Jun. 16, 2004; and German application 10 2004 062
`543.3 filed Dec. 24, 2004.
`
`SUBMISSION ON COMPACT DISC
`
`The contents of the following submission on compact
`discs are incorporated herein by reference in it s entirety:
`two copies of the Sequence Listing (COPY 1 and COPY 2)
`and a computer readable form copy of the Sequence Listing
`(CRF COPY), all on compact disc, each containing: file
`name: “Sequence Listing-13987-00020-US”, date recorded:
`May 9, 2007, size: 613 KB.
`
`FIELD OF THE INVENTION
`
`The present invention relates to a process for the produc-
`tion of polyunsaturated fatty acids in the seed of transgenic
`plants by introducing, into the organism, nucleic acids which
`encode polypeptides with (n3-desaturase, A12-desaturase,
`A6-desaturase, A6-elongase, A5-desaturase, A5-elongase
`and/or A4-desaturase activity, preferably polypeptides with
`A6-desaturase, A6-elongase and A5-desaturase activity.
`The nucleic acid sequences are the sequences shown in
`SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 193, SEQ
`ID NO: 197, SEQ ID NO: 199 and SEQ ID NO: 201.
`Preferably, a further nucleic acid sequence which encodes a
`polypeptide with a A12-desaturase activity is additionally
`introduced into the plant, in addition to these nucleic acid
`sequences, and also expressed simultaneously. Especially
`preferably, this is the nucleic acid sequence shown in SEQ
`ID NO: 195.
`
`These nucleic acid sequences can advantageously be
`expressed in the organism,
`if appropriate together with
`further nucleic acid sequences which encode polypeptides of
`the biosynthesis of the fatty acid or lipid metabolism.
`Especially advantageous are nucleic acid sequences which
`encode a A6-desaturase, a A5-desaturase, A4-desaturase,
`A12-desaturase and/or A6-elongase activity. These desatu-
`rases and elongases originate advantageously from Thalas-
`siosira, Euglena or Oslreococcus. Furthermore, the inven-
`tion relates to a process for the production of oils and/or
`triacylglycerides with an elevated content of long-chain
`polyunsaturated fatty acids.
`the invention furthermore
`In a preferred embodiment,
`relates to a process for the production of arachidonic acid,
`eicosapentaenoic acid or docosahexaenoic acid and to a
`process for the production of triglycerides with an elevated
`content of unsaturated fatty acids, in particular arichidonic
`acid, eicosapentaenoic acid and/or docosahexaenoic acid, in
`transgenic plants, advantageously in the seed of the trans-
`genic plant. The invention relates to the generation of a
`transgenic plant with an elevated content of polyunsaturated
`fatty acids, in particular arichidonic acid, eicosapentaenoic
`acid and/or docosahexaenoic acid, as the result of the
`
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`expression of the elongases and desaturases used in the
`process according to the invention.
`The invention furthermore relates to recombinant nucleic
`
`acid molecules comprising the nucleic acid sequences which
`encode the polypeptides with A6-desaturase, A6-elongase,
`A5-desaturase and A5-elongase activity, either jointly or
`individually, and transgenic plants which comprise the
`abovementioned recombinant nucleic acid molecules.
`
`A further part of the invention relates to oils, lipids and/or
`fatty acids which have been produced by the process accord-
`ing to the invention, and to their use. Moreover, the inven-
`tion relates to unsaturated fatty acids and to triglycerides
`with an elevated content of unsaturated fatty acids and to
`their use.
`
`DESCRIPTION OF RELATED ART
`
`the
`Lipid synthesis can be divided into two sections:
`synthesis of fatty acids and their binding to sn-glycerol-3-
`phosphate, and the addition or modification of a polar head
`group. Usual lipids which are used in membranes comprise
`phospholipids, glycolipids, sphingolipids and phosphoglyc-
`erides. Fatty acid synthesis starts with the conversion of
`acetyl-CoA into malonyl-CoA by acetyl-CoA carboxylase or
`into acetyl-ACP by acetyl transacylase. After condensation
`reaction,
`these two product molecules
`together
`form
`aceto