7
`.
`.
`5,614,393 A
`Inventors: Petra Cirpus, Mannheim (DE); Jorg
`6,043,411 A
`Bauer, Ludwigshafen (DE); Xiao Qiu,
`6,459,018 Bl
`Saskatoon (CA); Guohai Wu,
`6,884,921 B2
`Ssotoon CA}, Nagamanl Dail OE BY
`Saskatoon (CA)
`2004/0053379 Al
`2004/0111763 Al
`2004/0172682 Al*
`2008/0155705 Al
`2009/0222951 Al
`2010/0021976 Al
`
`.
`:
`(73) Assignee: BASF Plant Science GmbH,
`Ludwigshafen (DE)
`
`(*) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 1278 days.
`
`3/1997 Thomasetal.
`3/2000 Nishizawaetal.
`10/2002 Knutzon
`4/2005 Browseetal.
`NaNCardIc.
`et al.
`3/2004. Lerchlet al.
`6/2004 Heinzetal.
`9/2004 Kinney et al. vcesccsseeee 800/281
`6/2008 Zanketal.
`9/2009 Cirpuset al.
`1/2010 Lerchl et al.
`
`US009458436B2
`
`a2) United States Patent
`US 9,458,436 B2
`(10) Patent No.:
`
` Cirpusetal. (45) Date of Patent: *Oct. 4, 2016
`
`
`(54) METHOD FOR PRODUCING
`POLYUNSATURATED FATTY ACIDS IN
`TRANSGENIC PLANTS
`
`(56)
`
`References Cited
`US. PATENT DOCUMENTS
`“
`
`(75)
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21) Appl. No.:
`:
`
`10/590,457
`
`(86) PCT No.:
`
`PCT/EP2005/001863
`
`§ 371 ©)(),
`(2), (4) Date: Aug. 25, 2006
`(87) PCT Pub. No.: WO2005/083093
`PCT Pub. Date: Sep. 9, 2005
`
`CA
`DE
`DE
`EP
`EP
`WO
`
`WO
`WO
`WO
`wo
`wo
`
`FOREIGN PATENT DOCUMENTS
`
`2 485 060
`101 02 337 Al
`102 19 203
`0 550 162
`0 794 250
`WO-91/13972
`
`11/2003
`7/2002
`11/2003
`7/1993
`9/1997
`9/1991
`
`WO-93/11245
`WO-94/11516
`WO-94/18337
`WO-95/18222 Al
`WO-96/21022
`
`6/1993
`5/1994
`8/1994
`7/1995
`7/1996
`Continued
`(Continued)
`OTHER PUBLICATIONS
`
`(65)
`
`(30)
`
`Prior Publication Data
`US 2009/0222951 Al
`Sep. 3, 2009
`
`GeneSeq Accession ABV74261, Lerchl et al (Mar. 28, 2003).*
`(Continued)
`
`Foreign Application Priority Data
`
`Feb. 27, 2004
`Mar. 13, 2004
`Apr. 8, 2004
`May 14, 2004
`Jul. 16, 2004
`Dec. 24, 2004
`
`(DE) wo. 10 2004 009 457
`see 10 2004 012 370
`
`(DB) ......
`see 10 2004 017 518
`
`(DB)......
`beseeeeeeees 10 2004 024 014
`
`(DE)......
`bese PCT/EP2004/007957
`
`(EP) .......
`beseeeeeeees 10 2004 062 543
`
`(DB)......
`
`(51)
`
`Int. Cl.
`
`CI2N 15/82
`CI2N 9/02
`A23D 9/00
`AGIK 8/92
`A61Q 19/00
`CIN 9/10
`AGIK 8/36
`AGIK 31/202
`U.S. Cl.
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`CPC veeceecssseen C12N 9/0071 (2013.01); A23D 9/00
`(2013.01); A61K 8/361 (2013.01); A6LK 8/922
`(2013.01); A61K 31/202 (2013.01); A6IQ
`19/00 (2013.01); CI2N 9/0083 (2013.01);
`CI2N 9/1029 (2013.01); CI2N 15/8247
`(2013.01); A61K 2800/86 (2013.01); CI12Y
`114/19 (2013.01)
`
`Field of Classification Search
`None
`
`(52)
`
`(58)
`
`Primary Examiner — Elizabeth McElwain
`(74) Attorney, Agent, or Firm — 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 m3-desaturase, Al2-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 tooils, 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
`
`Page 2
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`(56)
`
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`CSIRO Exhibit 1013
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`

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`3 of 290
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`CSIRO Exhibit 1013
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`U.S. Patent
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`Oct. 4, 2016
`
`Sheet 1 of 33
`
`US 9,458,436 B2
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`U.S. Patent
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`Oct. 4, 2016
`
`Sheet 2 of 33
`
`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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`Oct. 4, 2016
`
`Sheet 3 of 33
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`US 9,458,436 B2
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`Figure 3:
`
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`6 of 290
`6 of 290
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`U.S. Patent
`
`Oct. 4, 2016
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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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`o“A
`
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`26:0
`(DHA)
`
`Retention time
`
`=&a
`
`a
`re
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`7 of 290
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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 pYes3-OmELO3/pYes2-EgD4or pYes3-
`OmELO3/pYes2-EgD4+pESCLeu-PtD5. The yeast cells were cultured in
`minimal medium without tryptophan and uracil/ and leucin in the presence
`of 250 pM 20:5°5%11-1417 and 18:44°°12'5respectively. 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) + standard deviation.
`
`pYes3-OmELO/pYes2-EgD4
`
`pYes3-OmELO/pYes2-EgD4 EgD4
`+ pESCLeu-PtD5
`
`Fatty acids
`
`Feeding of 20:545%111417
`
`Feeding of 18:445%'215
`
`16:0
`
`16:1
`
`18:0
`
`18:1
`
`18:14"
`
`9.35 + 1.61
`
`14.70 + 2.72
`
`5.11 + 1.09
`
`19.49 + 3.01
`
`18.93 + 2.71
`
`18:4 469.12:15
`
`-
`
`20:14"
`
`20:14"
`
`20:448101417
`
`20:5°581114617
`
`22:4A10131617
`
`22:5 A71013:16,19
`
`22:6 24071013,16,19
`
`3.24 40.41
`
`11.134 2.07
`
`-
`
`6.91+ 1.10
`
`-
`
`8.77 + 1.32
`
`2.73 + 0.39
`
`7.35 1.37
`
`10.02 + 1.81
`
`4.27+1.21
`
`10.81 + 1.95
`
`11.61 + 1.48
`
`7.79 + 1.29
`
`1.56 + 0.23
`
`4.40 + 0.78
`
`30.05 + 3.16
`
`3.72 + 0.59
`
`5.71 + 1.30
`
`1.10 + 0.27
`
`0.58 + 0.10
`
`8 of 290
`8 of 290
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`U.S. Patent
`
`Oct. 4, 2016
`
`Sheet 6 of 33
`
`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
`
`US 9,458,436 B2
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`10 of 290
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`CSIRO Exhibit 1013
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`

`U.S. Patent
`
`Oct. 4, 2016
`
`Sheet 8 of 33
`
`US 9,458,436 B2
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`11 of 290
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`

`U.S. Patent
`
`Oct. 4, 2016
`
`Sheet 9 of 33
`
`US 9,458,436 B2
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`12 of 290
`12 of 290
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`Oct. 4, 2016
`
`Sheet 10 of 33
`
`US 9,458,436 B2
`
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`13 of 290
`13 of 290
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`U.S. Patent
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`Oct. 4, 2016
`
`Sheet 11 of 33
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`US 9,458,436 B2
`
`
`
`
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`14 of 290
`14 of 290
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`U.S. Patent
`
`Oct. 4, 2016
`
`Sheet 12 of 33
`
`US 9,458,436 B2
`
`Figure 12: Desaturation ofy-linolenic acid (18:2 w6-fatty acid) to give a-linolenic acid
`(18:3 w3-fatty acid) by Pi-omega3Des.
`
`piOMEGA3 + 18:2
`
`15 of 290
`15 of 290
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`Oct. 4, 2016
`
`Sheet 13 of 33
`
`US 9,458,436 B2
`
`Figure 13: Desaturation of y-linolenic acid (18:2 w6-fatty acid) to give stearidonic acid
`(18:4 w3-fatty acid) by Pi-omega3Des.
`
`pIOMEGAS + y-18:3
`
`16 of 290
`16 of 290
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`Oct. 4, 2016
`
`Sheet 14 of 33
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`US 9,458,436 B2
`
`Figure 14: Desaturation of C20:2 w6-fatty acid to give C20:3 3-fatty acid by Pi-
`omega3Des.
`
`piOMEGA3 + C20:2411*
`
`17 of 290
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`Oct. 4, 2016
`
`Sheet 15 of 33
`
`US 9,458,436 B2
`
`Figure 15: Desaturation of C20:3 w6-fatty acid to give C20:4 w3-fatty acid by Pi-
`omega3Des.
`
`piOMEGA3 + C20:3°2'1-"4
`
`18 of 290
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`Oct. 4, 2016
`
`Sheet 16 of 33
`
`US 9,458,436 B2
`
`Figure 16: Desaturation of arachidonic acid (C20:4 w6-fatty acid) to give eicosapen-
`taenoic acid (C20:5 w3-fatty acid) by Pi-omega3Des.
`
`piOMEGA3 + C20:445:811,14
`
`19 of 290
`19 of 290
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`Oct. 4, 2016
`
`Sheet 17 of 33
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`US 9,458,436 B2
`
`Figure 17: Desaturation of docosatetraencic acid (C22:4 w6-fatty acid) to give
`docosapentaenocic acid (C22:5 w3-fatty acid) by Pi-omega3Des.
`
`piOMEGAS + 22:4
`
`20 of 290
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`Oct. 4, 2016
`
`Sheet 18 of 33
`
`US 9,458,436 B2
`
`
`
`oO
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`21 of 290
`21 of 290
`
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`

`U.S. Patent
`
`Oct. 4, 2016
`
`Sheet 19 of 33
`
`US 9,458,436 B2
`
`
`
`Fraction
`
`© ©D
`
`d)
`
`®EO o
`
`Figure19:Desaturationofphospholipid-boundarachidonicacidtogiveEPAbyPi-Omega3Des
`
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`PSJEINJESSP P-02 %
`
`22 of 290
`22 of 290
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`Oct. 4, 2016
`
`Sheet 20 of 33
`
`US 9,458,436 B2
`
`Figure 20: Conversion oflinoleic acid (arrow) to give y-linolenic acid (y-18:3) by Ot-
`Des6.1.
`
`Absorption mAU
`
`16:0 16:1
`
`Retention time
`
`23 of 290
`23 of 290
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`
`Oct. 4, 2016
`
`Sheet 21 of 33
`
`US 9,458,436 B2
`
`Figure 21: Conversion oflinoleic 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 OtD6.1.
`
`A) OtD6+LA
`
`GLA
`
`LA
`
`B) OtD6+PSE1+PtD5+LA
`
`
`
`C) OtD6+ALA
`
`ALA
`
`| <+— STA
`
`D) OtD6+PSE1+PtD5+ALA
`
`24 of 290
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`Oct. 4, 2016
`
`Sheet 22 of 33
`
`US 9,458,436 B2
`
`Figure 22: Expression of ELO(XI) in yeast
`
`Absorption in mA
`
`A) ELO (XI!) without fatty acid feeding
`B) ELO (XI) + 18:4A6,9,12,15 (250 yM)
`C) ELO(XI) + 20:5 (500 yM) Retention time in min
`
`25 of 290
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`Oct. 4, 2016
`
`Sheet 23 of 33
`
`US 9,458,436 B2
`
`Figure 23:
`
`Absorption in mA
`
`A) EIO(Ci) withoutfatty acid feeding
`B) ELO (Ci) + 18:4 (250 uM)
`C) ELO (Ci) + 20:5 (500 yM) Retention time in minutes
`
`26 of 290
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`Oct. 4, 2016
`
`Sheet 24 of 33
`
`US 9,458,436 B2
`
`Figure 24: Elongation of eicosapentaenoic acid by OtElo1 (B) and OtElo1.2 (D), re-
`spectively. The controls (A, C) do not show the elongation product (22:53).
`
`FIDSignal
`
`18:149
`
`
`
`20:545.8,11,14,17
`
`
`
`22:57,10,13,16,19
`20:5.45.8,11,14,17
`
`23
`
`275
`
`30
`
`325
`
`35
`
`37.5
`
`a0
`
`425
`
`45
`
`min
`
`Retention time (min)
`
`20:503
`
`pYES2
`
`
`
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`Oct. 4, 2016
`
`Sheet 25 of 33
`
`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 (22:4a6).
`
`FIDSignal
`
`
`
`20:4458,11,14
`
`
`22:4A7,10,13,16
`
`—
`20:4.45,8,11,14
`
`
`Retention time (min)
`
`
`
`28 of 290
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`Oct. 4, 2016
`
`Sheet 26 of 33
`
`US 9,458,436 B2
`
`Figure 26: Elongation of 20:5n-3 by the elongases At3g06470.
`
`Absorption in mA
`
`20:5(n-3)
`
`22:5(n-3)
`
`13
`
`15
`
`17
`
`19
`
` 5
`
`7
`
`9
`
`11
`
`Retention time in minutes
`
`29 of 290
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`Oct. 4, 2016
`
`Sheet 27 of 33
`
`US 9,458,436 B2
`
`Figure 27:
`
`Substrate specificity of the Xenopus Elongase (A), Ciona Elongase (B) und
`Oncorhynchus Elongase (C)
`
`A)
`
`B)
`
`C)
`
`
`
`
`
`absoluteelongation(%)
`
`20
`
`15
`
`10
`
`25
`
`20
`
`15
`
`10
`
`60
`
`45
`
`30
`
`15
`
`100
`
`75
`
`50
`
`25
`
`100
`
`75
`
`50
`
`25
`
`100
`
`75
`
`50
`
`25
`
`
`
`
`
`relativeelongation(%)
`
`
`
`
`
`
`
`\ jj 48jyAIT)Ag N A6 A5 |
`
`
`
`
`
`
`
`18:2w6
`
`18:3w3 $3 8
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`
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`8
`
`20:3w3
`
`20:4w6
`
`20:5w3
`
`22:6w3
`
`9
`&
`
`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 A6-elongase (D)
`
`A)
`
`B)
`
`C)
`
`D)
`
`
`
`
`
`absoluteelongation(%)
`
`Fatty Acid
`
`60
`
`45
`
`30
`
`15
`
`0
`
`25
`
`20
`
`15
`
`10
`
`45
`
`10
`
`60
`
`40
`
`20
`
`100
`
`7
`
`50
`
`95
`
`100
`
`75
`
`50
`
`25
`
`100
`
`75
`
`50
`
`25
`
`100
`
`75
`
`50
`
`25
`
`
`
`
`
`relativeelongation(%)
`
`EOSSF
`A048ooswa|
`18:206)
`18:3w6
`18:4w3
`18:3w3
`20:3w
`20:3
`
`22:6w3
`
`31 of 290
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`Oct. 4, 2016
`
`Sheet 29 of 33
`
`US 9,458,436 B2
`
`Figure 29:
`
`Expression of the Phaeodactylum tricornutum A6-elongase (PtELO6)in
`yeast. A) shows the elongation of the C18:3°°°""? fatty acid and B) the
`elongation of the C18:34°*'2:"5 fatty acid
`
`A)
`
`B)
`
`18:1°°
`
`493.456.9,121,15
`
`32 of 290
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`Oct. 4, 2016
`
`Sheet 30 of 33
`
`US 9,458,436 B2
`
`Figure 30:
`
`Figure 30 showsthe substrate specificity of PtELO6 with regard to the sub-
`strates fed.
`
`PtELO6specificity
`
`
`
`Elongation(%)
`
`(1sn
`
`
`
`80
`
`60
`
`40
`
`20
`
`0
`
`18:2
`
`a18:3
`
`18:3
`
`18:4
`
`Fatty acid substrate
`
`33 of 290
`33 of 290
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`U.S. Patent
`
`Oct. 4, 2016
`
`Sheet 31 of 33
`
`US 9,458,436 B2
`
`
`_;_.(Z1‘6‘9VE:81)
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`CSIRO Exhibit 1013
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`U.S. Patent
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`Oct. 4, 2016
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`Sheet 32 of 33
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`US 9,458,436 B2
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`CSIRO Exhibit 1013
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`U.S. Patent
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`Oct. 4, 2016
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`Sheet 33 of 33
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`CSIRO Exhibit 1013
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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
`US.C. 371) of PCT/EP2005/001863filed 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 COPY2)
`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 m3-desaturase, Al2-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 Ostreococcus. 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 docosahexaenoicacid, 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 furtherpart 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-ACPby acetyl transacylase. After condensation
`reaction,
`these two product molecules
`together
`form
`acetoacetyl-ACP, which is c