United States Patent
`US 10,301,638 B2
`0) Patent No.:
`(12)
`Cirpusetal.
`(45) Date of Patent:
`May28, 2019
`
`
`US010301638B2
`
`(54) OILS, LIPIDS AND FATTY ACIDS
`PRODUCED IN TRANSGENIC BRASSICA
`PLANT
`
`(72)
`
`:
`.
`:
`(71) Applicant: BASF Plant Science GmbH,
`Ludwigshafen (DE)
`.
`.
`,
`Inventors: Petra Cirpus, Mannheim (DE); Jorg
`Bauer, Limburgerhof (DE); Xiao Qiu,
`Saskatoon (CA); Guohai Wu,
`Saskatoon (CA); Bifang Cheng,
`Saskatoon (CA); Martin Truksa,
`Saskatoon (CA); Tom Wetjen,
`Mannheim (DE)
`(73) Assignee: BASF Plant Science GmbH,
`:
`Ludwigshafen (DE)
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(*) Notice:
`
`4/2005 Browseet al.
`6,884,921 B2
`5/2007 Mukerji etal.
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`7/2007 Kang
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`11/2011 Cirpus et al.
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`1/2012 Lerchl et al.
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`soonolees ‘i
`.04 Rae et a
`inney et al.
`2008/0076164 Al
`3/2008. Cirpusetal.
`2008/0155705 Al
`6/2008 Zank et al.
`(Continued)
`FOREIGN PATENT DOCUMENTS
`
`8/2001
`2001239244 B2
`AU
`(21) Appl. No.: 15/256,914
`
`: 2003232512 B2=11/2003AU
`
`(22)
`Filed:
`Sep. 6, 2016
`(Continued)
`(65)
`Prior Publication Data
`
`OTHER PUBLICATIONS
`
`US 2016/0369290 Al
`
`Dec. 22, 2016
`
`Related U.S. Application Data
`
`(63) Continuation of application No. 12/280,090, filed as
`application No. PCT/EP2007/051675 on Feb. 21,
`2007.
`
`(30)
`
`Foreign Application Priority Data
`
`Feb. 21, 2006
`Sep. 7, 2006
`
`(DE) wee 10 2006 008 030
`(EP) wceeeeesssesereeecensenees 06120309
`
`U.S. Appl. No. 60/613,861, Singh et al.
`(Continued)
`
`Primary Examiner — Hope Robinson
`(74) Altorney, Agent, or Firm — Drinker Biddle & Reath
`LLP
`
`(57)
`
`ABSTRACT
`
`(51)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`Int. Cl.
`The invention relates to a method for producing eicosapen-
`AGIK 31/20
`taenoic acid, docosapentaenoic acid and/or docohexanoic
`CI2N 15/82
`acid in transgenic plants. According to said method, the
`C1I2N 9/02
`plant is provided with at least one nucleic acid sequence
`CLIN 10
`coding for a polypeptide with a A6 desaturase activity, at
`CI2N 15/52
`least one nucleic acid sequence coding for a polypeptide
`C12P 7/64
`with a A6 elongase activity, at
`least one nucleic acid
`(52) U.S. Cl.
`sequence coding for a polypeptide with a A5 desaturase
`CPC ....... CI2N 15/8247 (2013.01); C12N 9/0071
`(2013.01); C12N 9/0083 (2013.01); CI2N_activity, and at least one nucleic acid sequence coding for a
`9/1029 (2013.01); C12N 15/52 (2013.01):
`polypeptide with a A5 elongase activity, the nucleic acid
`C12P 7/6427 (2013.01), C12Y 114/19
`sequence coding for a polypeptide with a A5 elongase
`(2013.01)
`activity being modified in relation to the nucleic acid
`sequence in the organism from which the sequence origi-
`nates, such that it is adapted to the codonusein at least one
`type of plant. For the production of docosahexanoicacid,at
`least one nucleic acid sequence coding for a polypeptide
`with a A4 desaturase activity is also introduced into the
`plant.
`
`(58) Field of Classification Search
`.
`None
`See application file for complete search history.
`:
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`

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`(56)
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`US 10,301,638 B2
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`1
`OILS, LIPIDS AND FATTY ACIDS
`PRODUCED IN TRANSGENIC BRASSICA
`PLANT
`
`RELATED APPLICATIONS
`
`This application is a continuation of patent application
`Ser. No. 12/280,090 filed Aug. 20, 2008, which is a national
`stage application (under 35 U.S.C. § 371) of PCT/EP2007/
`051675, filed Feb. 21, 2007, which claims benefit of German
`application 10 2006 008 030.0, filed Feb. 21, 2006 and
`European application 06120309.7, filed Sep. 7, 2006. The
`entire content of each aforementioned application is hereby
`incorporated by reference in its entirety.
`
`SUBMISSION OF SEQUENCE LISTING
`
`The Sequence Listing associated with this application is
`filed in electronic format via EFS-Web and hereby incorpo-
`rated by reference into the specification in its entirety. The
`name of the text file containing the Sequence Listing is
`Sequence_Listing074017_0013_01. The size of the text
`file is 730 KB, andthetext file was created on Sep. 2, 2016.
`The present invention relates to a process for the produc-
`tion of eicosapentaenoic acid, docosapentaenoic acid and/or
`docosahexaenoic acid in transgenic plants, providing in the
`plant at least one nucleic acid sequence which codes for a
`polypeptide having a A6-desaturase activity; at least one
`nucleic acid sequence which codes for a polypeptide having
`a A6-elongase activity; at least one nucleic acid sequence
`which codes for a polypeptide having a A5-desaturase
`activity; and at least one nucleic acid sequence which codes
`for a polypeptide having a A5-elongase activity, where the
`nucleic acid sequence which codes for a polypeptide having
`a A5-elongase activity is modified by comparison with the
`nucleic acid sequence in the organism from which the
`sequenceis derived in that it is adapted to the codon usage
`in one or more plant species.
`In a preferred embodimentthere is additionally provision
`of further nucleic acid sequences which code for a polypep-
`tide having the activity of an w3-desaturase and/or of a
`A4-desaturase in the plant.
`In a further preferred embodiment there is provision of
`further nucleic acid sequences which code for acyl-CoA
`dehydrogenase(s), acyl-ACP (acyl carrier protein) desatu-
`rase(s),
`acyl-ACP
` thioesterase(s),
`fatty
`acid
`acyl
`transferase(s),
`acyl-CoA:lysophospholipid
`acyl
`transferase(s),
`fatty
`acid
`synthase(s),
`fatty
`acid
`hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-
`coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid
`acetylenases, lipoxygenases, triacylglycerol lipases, allene
`oxide
`synthases, hydroperoxide lyases or
`fatty acid
`elongase(s) in the plant.
`The invention furthermore relates to recombinant nucleic
`
`least one nucleic acid
`acid molecules comprising at
`sequence which codes for a polypeptide having a A6-de-
`saturase activity; at least one nucleic acid sequence which
`codes for a polypeptide having a A5-desaturase activity; at
`least one nucleic acid sequence which codes for a polypep-
`tide having a A6-elongase activity; and at least one nucleic
`acid sequence which codes for a polypeptide having a
`A5-elongase activity and which is modified by comparison
`with the nucleic acid sequence in the organism from which
`the sequence originates in that it is adapted to the codon
`usage in one or more plant species.
`
`10
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`15
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`20
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`25
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`30
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`40
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`45
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`50
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`55
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`60
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`65
`
`2
`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.
`Finally,
`the invention also relates to transgenic plants
`which have been producedby the process of the invention or
`which comprise a recombinant nucleic acid molecule of the
`invention, and to the use thereof as foodstuffs or feedstuffs.
`Lipid synthesis can be divided into two sections:
`the
`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 converted via a series of conden-
`sation, reduction and dehydration reactions so that a satu-
`rated fatty acid molecule with the desired chain length is
`obtained. The production of the unsaturated fatty acids from
`these molecules is catalyzed by specific desaturases, either
`aerobically by means of molecular oxygen or anaerobically
`(regarding the fatty acid synthesis in microorganisms,see F.
`C. Neidhardt et al. (1996) F. coli and Salmonella. ASM
`Press: Washington, D.C., p. 612-636 and references cited
`therein; Lengeleret al. (Ed.) (1999) Biology of Procaryotes.
`Thieme: Stuttgart, New York, and the references therein, and
`Magnuson, K., et al.
`(1993) Microbiological Reviews
`57:522-542 and the references therein).