ES2653727T3 - Desaturasas y proceso para la producción de ácidos grasos poliinsaturados en organismos transgénicos - Google Patents

Desaturasas y proceso para la producción de ácidos grasos poliinsaturados en organismos transgénicos Download PDF

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ES2653727T3
ES2653727T3 ES13180237.3T ES13180237T ES2653727T3 ES 2653727 T3 ES2653727 T3 ES 2653727T3 ES 13180237 T ES13180237 T ES 13180237T ES 2653727 T3 ES2653727 T3 ES 2653727T3
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acid sequence
nucleic acid
production
desaturases
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Toralf Senger
Jörg BAUER
Johnathan A. Napier
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BASF Plant Science GmbH
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    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6431—Linoleic acids [18:2[n-6]]
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6432—Eicosapentaenoic acids [EPA]
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6434—Docosahexenoic acids [DHA]
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
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    • C12P7/6481—Phosphoglycerides
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
    • C12Y114/19001—Stearoyl-CoA 9-desaturase (1.14.19.1), i.e. DELTA9-desaturase

Abstract

Un polinucleótido que comprende una secuencia de ácidos nucleicos seleccionada del grupo que consiste de: (a) una secuencia de ácidos nucleicos como se muestra en la SEQ ID NO. 50 o 51; (b) una secuencia de ácidos nucleicos que codifica un polipéptido que caracteriza una secuencia de aminoácidos como se muestra en cualquiera de la SEQ ID NO. 52; (c) una secuencia de ácidos nucleicos que codifica un polipéptido con una secuencia de aminoácidos con por lo menos 75 % de identidad con la SEQ ID NO. 52, y que codifica un polipéptido con una actividad de omega-3- desaturasa; y (d) una secuencia de ácidos nucleicos obtenible u obtenida mediante la amplificación de ácido nucleico del material genético de Hyaloperonospora parasitica usando el primer par - SEQ ID NO. 53 y SEQ ID NO. 55, o - SEQ ID NO. 54 y SEQ ID NO. 56 y que codifica un polipéptido con una actividad de omega-3-desaturasa.

Description

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Tabla 4A: Alimentación de levaduras
Nombre de la muestra/ carga de ácido graso
Etapa de reacción observada Índice de conversión (%) Actividad observada Fig.
Sustrato (% de contenido de ácido graso total)
Producto (% de contenido de ácido graso total) Esperado observado
Vector vacío pYES2 / 18: 2n-9
18:2n-6 25,3 18:3n-3 0,0 - - - 4a
d15Des (Ch)/18: 2n-9
18:2n-6 6,3 18:3n-3 5,7 > 0 47,5 ∆15-des. 4b
d15Des (Cy)/18: 2n-9 ω3Des (Hp)/18: 2n-9 ω3Des (Hp)/20: 3n-6 ω3Des (Hp)/20: 4n-6
18:2n-6 18:2n-6 20:3n-6 20:4n-6 13,0 12,6 6,3 5,7 18:3n-3 18:3n-3 20:4n-3 20:5n-3 0,9 0,0 0,6 1,8 > 0 > 0 > 0 > 0 6,4 0,0 8,6 24,0 ∆15-des. -ω3-Des ω3-Des 4c
A modo de control para el ensayo para la actividad ∆ 15-desaturasa, las levaduras se transforman con el vector vacío pYES, se carga el ácido graso 18:2 n-6 y el perfil de ácidos grasos se analiza (Figura 2A). En comparación, el 5 ácido graso adicional 18:3 n-3 se puede observar en levaduras que expresan las desaturasas d15Des (Ch) y d15Des(Cy) (Figura 2B, 2C). Estas desaturasas que se han ensayado, por lo tanto, tienen actividad ∆15-desaturasa.
Ejemplo 5: Producción de Plantas transgénicas para la producción de ácidos grasos poliinsaturados de cadena larga
Para producir ácidos grasos poliinsaturados de cadena larga en plantas, diversos genes de la ruta metabólica se combinan en un vector binario. Para producir el ácido graso ácido eicosapentaenoico (20:5∆5,8,11,14,17), se 10 combinan los genes como se describe en la Tabla 5. Análogamente, Los genes como se describe en la Tabla 6 se combinan para producir el ácido graso ácido docosahexaenoico (22:06∆4,7,10,13,16,19).
Tabla 5: Combinación de genes para la producción de ácido eicosapentaenoico
Gen
Actividad SEQ ID NO.
D6Des(Pir)
∆6-desaturasa 22
D6Elo(Pp)
∆6-elongasa 31
D5Des(Tc)
∆5-desaturasa 25
ω3-Des(Pi)
ω3-desaturasa 28
D15Des(Ch)
∆12-desaturasa 2
D15Des(Cy)
∆15-desaturasa 9
D12Des(Mac)
∆12-/∆15-desaturasa 16
ω 3Des(Hp)
ω3-desaturasa 51
35
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Claims (1)

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ES13180237.3T 2008-12-12 2009-12-08 Desaturasas y proceso para la producción de ácidos grasos poliinsaturados en organismos transgénicos Active ES2653727T3 (es)

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EP (2) EP2376638B1 (es)
AU (2) AU2009326129B2 (es)
CA (1) CA2744930C (es)
DE (1) DE112009003708T5 (es)
ES (2) ES2435572T3 (es)
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