New opportunities for oilseeds using to plants. Alternative ‰3-PUFA ... Biodegradable...

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Transcript of New opportunities for oilseeds using to plants. Alternative ‰3-PUFA ... Biodegradable...

  • New opportunities foroilseeds using biotechnology

    Allan GreenCSIRO Plant Industry

  • First wave Input Traits

    IP3

    Traits that improve crop production

    Stresstolerance

    Diseaseresistance

    Herbicidetolerance

    Insectresistance

    Perception that the benefits accrue only to growers and agribusiness

    - no consumer benefits!

    Hybridvarieties

  • Second Wave Output Traits

    Silencing endogenous genesHigh-oleic oilseeds

    Introducing new genes (transgenes)Oilseeds containing 3 fatty acids (EPA &DHA)Oilseeds producing industrial raw materials

  • Silencing genes in plantsHigh-oleic cooking oils

  • Properties of seed oil fatty acids

    1512916:0

    palmitic18:0stearic

    18:3linolenic

    18:2linoleic

    18:1oleic

    PUFASATURATES MUFA

    Stable Unstable

    LDL LDL cholesterol

    Essential fatty acids

    neutral

    stable & healthycooking oils

  • Hydrogenationhydrogenation

    higher m.p. fatty acids

    unstable polyunsaturates

  • Hydrogenationhydrogenation

    additional cost

    trans fatty acids are nutritionally undesirable

  • Hydrogenationhydrogenation

    additional cost

    trans fatty acids are nutritionally undesirable

  • Tailoring composition to use

    High-stearic

    High-oleicGene technology enables oil composition to be redesigned by

    mutating or silencing genes controlling fatty acid synthesis

    High-oleic cooking oils from most oilseeds

  • High-oleic sunflower oilInducing gene mutations

    Chemically-inducedmutations (EMS)

    12-desaturasemutations

    16:0 18:0 18:1 18:2 18:3

    Sunflower oil 297 -604

    High-oleic sunflower (Sunola) 847 -54

  • Cottonseed oil composition

    16:0palmitic

    75

    50

    25

    0 18:2linoleic

    18:1oleic

    18:0stearic 9 12

  • 12-desaturase silencing

    Preventing RNA translation (PTGS)

    12-desaturaseir-DNA

    Complete coding region of targetgene with 5 inverted repeat (850nt)

    18:116:0 18:318:218:0

    1525 -572Cottonseed oil

    7817 -41High-oleic cottonseed oil

  • High-oleic cottonseed oil

    16:0palmitic

    18:0stearic

    18:2linoleic

    18:1oleic

    Gene silencing12-desaturase

    12

    75

    50

    25

    0

    HO-CSO forcommercial

    frying

  • 9-desaturase silencing

    Preventing RNA translation (PTGS)

    9-desaturaseir-DNA

    Complete coding region of targetgene with 5 inverted repeat (580nt)

    18:116:0 18:318:218:0

    1525 -572Cottonseed oil

    515 -3840High-stearic cottonseed oil

  • High-stearic cottonseed oil

    16:0palmitic

    75

    50

    25

    0

    Gene silencing9-desaturase

    HS-CSO formargarinehardstock

    18:2linoleic

    18:0stearic

    18:1oleic9

  • Novel CSO fatty acid profiles

    Fatty acid composition (%)

    Palmitic Stearic Oleic Linoleic

    Coker 315 26 2 15 57

    High oleic 17 1 78 4

    High stearic 15 40 5 38

  • Novel CSO fatty acid profiles

    Fatty acid composition (%)

    Palmitic Stearic Oleic Linoleic

    Coker 315 26 2 15 57

    High oleic 17 1 78 4

    High stearic 15 40 5 38

    Combined 14 23 54 7

  • Availability of high-oleic oilsCommercially

    availableTechnically

    availableOleic(%)Type

    cottonseed GM 78 ?

    safflower Non-GM 81sunflower Non-GM 75,85

    peanut Non-GM 76canola Non-GM 60-70canola GM 88 ?

    soybean GM 84 ?

  • Adding genes to plantsOilseeds containing long

    chain 3 fatty acids

  • Health benefits of 3 consumption

    Reduced risk of cardiovascular disease & clottingImproved blood pressure regulation and platelet functionReduced risk of cancers such as prostate and bowelTreatment of rheumatoid arthritis and some forms of depressionImproved foetal and infant development

  • Long chain 6(n-6) and 3(n-3) PUFA

    EPA20:5

    DHA22:6

    SDA18:4

    22:5

    20:4

    18:318:218:118:0

    AA 20:4

    22:5

    GLA 18:3

    22:4

    20:3

    2 & 4 3 & 5 eicosanoids

  • Potential new sources of 3 oils

    Marine sources of 3 fatty acids are declining will not meet future global needsDietary sources can be expanded by transferring 3 biosynthetic pathway to plants

  • Alternative 3-PUFA pathways

    DHAEPAelongase

    5 desaturase

    elongase

    4 desaturase

    algae, mosses, fungi, nematodes

    algae, mosses, fungi, nematodes

    polyketide synthesisbacteria, thraustochytrids (anaerobic)

    18:46 desaturase

    Echium

    acetylCoA

    18:3

    DHAEPA18:4elongase (x2)

    6 desaturase

    -oxidation

    mammals

    18:3 6 desaturaseelongase

    5 desaturase

  • Engineering 3-PUFA in plants16:0

    palmitic

    18:2linoleic

    18:1oleic

    18:3linolenic

    18:0stearic

    61518:4SDA

    22:5

    20:4 20:5EPA

    22:6DHA

    SDA is efficientlyconverted to

    EPA & DHA in the human body

    Michael James & Les Cleland, Royal Adelaide Hospital

    Canola oil with 17% SDA has been developed by

    enhancement of 15 desaturase and addition of 6 desaturase genes

  • Engineering 3-PUFA in oilseeds

    EPA20:5

    SDA18:4

    20:4

    18:3

    DHA22:6

    22:55 elongase

    4 desaturase

    13kb cassette

    AA 20:4

    GLA 18:3

    20:3

    6 desaturase

    6 elongase

    5 desaturase

    8.5kb cassette

    18:0 18:1 18:2

  • Engineering 3-PUFA in yeast

    Exogenously supplied 18:3

    20:5, EPA

    Yeast expressing 6 & 5 desaturase and

    elongase genes

    Beaudoin et al. (2000) PNAS 97: 6421-6426

  • Engineering 3-PUFA in plants

    Exogenously supplied 22:5

    22:6, DHAB. juncea expressing a 4-desaturase gene

    Qiu et al. (2001) J. Biol. Chem. 276: 31561-31566

  • Other nutritional improvements

    THE FUTURE OF FOOD AND NUTRITION WITH BIOTECHNOLOGY

    Removing food allergens through silencing specific proteins P34 in soybeans

    Enhanced -carotene synthesis to overcome Vitamin A deficiency Golden Rice, canola oil

    Elevated levels of antioxidants increased levels of -tocopherol

    Increased mineral content iron in rice from Phaseolis ferretin

    protein

  • The market will decide

  • Adding genes to plantsRenewable sources of

    industrial raw materials

  • Industrial crops

    We can envisage processing factories being designed to specifically match up with the genetics in the crops

    Richard McConnell, Pioneer Hi-Bred

    Metabolic EngineeringGene technology be usedto dramatically alter the

    composition of plantproducts to create newindustrial raw materials

  • Natural diversity for chemicals

    genes fromwild plants

    crops

    renewableindustrial raw

    materials

  • Industrial fatty acids

    conjugated fatty acids(superior drying oils)

    petroselenic acid(polymers, detergents)

    ricinoleic acid(lubricants, cosmetics

    pharmaceuticals)

    vernolic acid(resins, coatings,

    plasticisers)

    erucic acid(polymers, cosmetics,inks, pharmaceuticals)

    lauric acid(detergents)

  • Lauric canola

    zero laurate laurate thioesterase genefrom California bay tree

    laurate acyltransferasegene from coconut

    40% laurate

    70% laurate

    TE

    LPAAT

    canola oil

  • Epoxy fatty acids

    Seeds of wild Crepis spp contain

    high contents of epoxy fatty acids

    Valuable chemicals used in glues, resins and surface coatings currently obtained from petrochemicals or

    processed vegetable oils

  • Increasing epoxy fatty acids0 70Epoxy fatty acids in seed oil (%)

    Fatty acid epoxygenase gene (Cpal2) cloned from Crepis palaestina

    Cpal2 Cpdes +++ Cpal2

    ??& silence competing enzymes

    Cpal2 + What limits accumulation?

  • Biodegradable plasticsC

    C

    C

    CC

    CHO

    OC

    Polyhydroxybutyrate (PHB)

    CC

    CO

    OC

    CC

    CO

    OC

    CC

    CO

    OC

    CC

    CO

    OC

    CC

    CHO

    OC

    CC

    CHO

    O

    C

    C

    C

    CC

    CHO

    O

    C

    C

    Biodegradable polymers based on a family of R-3 hydroxy-alkanoic acids

    3-OH-butyrate 3-OH-valerate 3-OH-caproate 3-OH-heptanoate

  • Biodegradable plasticsO

    CC

    CHO

    C

    CC

    CO

    OC

    CC

    CO

    OC

    CC

    CO

    OC

    CC

    CO

    OC

    CC

    CO

    OC

    CC

    O

    CO

    C

    +Aceto-acetyl-

    CoA reductase-keto

    thiolase

    PHB is synthesised from acetyl-CoA by action of only three enzymes

    PHB is synthesised from acetyl-CoA by action of only three enzymes

    PHB synthase

  • normal vigour

    Plastic plants

    Chloroplast Localisation Signal (CLS) added to target each

    enzyme to chloroplasts

    PHB = 14% (dry wt)

    IP3

    CLS ketothiolase

    CLS reductase

    CLS synthase

  • Industrial vs Food crops

    Industrial products produced from traditional food crops (e.g. oilseeds) will require strict segregation from food-grade products because they:-

    will not be approved for food usemay actually be toxic

    Genetic Isolation & Identity Preservation will be important crop and product management tools

  • Silencing gene expression

    Intron

    Splicing

    Gene

    mRNA

    Protein

    TP

    Intron Sequen