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New opportunities foroilseeds using biotechnology
Allan GreenCSIRO Plant Industry
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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
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Second Wave – Output Traits
Silencing endogenous genesHigh-oleic oilseeds
Introducing new genes (transgenes)Oilseeds containing ω3 fatty acids (EPA &DHA)Oilseeds producing industrial raw materials
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Silencing genes in plantsHigh-oleic cooking oils
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Properties of seed oil fatty acids
∆15∆12∆916:0
palmitic18:0stearic
18:3linolenic
18:2linoleic
18:1oleic
PUFASATURATES MUFA
Stable Unstable
LDL LDL cholesterol
Essential fatty acids
neutral
stable & healthycooking oils
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Hydrogenationhydrogenation
higher m.p. fatty acids
unstable polyunsaturates
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Hydrogenationhydrogenation
additional cost
trans fatty acids are nutritionally undesirable
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Hydrogenationhydrogenation
additional cost
trans fatty acids are nutritionally undesirable
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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
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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
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Cottonseed oil composition
16:0palmitic
75
50
25
0 18:2linoleic
18:1oleic
18:0stearic ∆9 ∆12
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∆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
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High-oleic cottonseed oil
16:0palmitic
18:0stearic
18:2linoleic
18:1oleic
Gene silencing∆12-desaturase
∆12
75
50
25
0
HO-CSO forcommercial
frying
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∆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
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High-stearic cottonseed oil
16:0palmitic
75
50
25
0
Gene silencing∆9-desaturase
HS-CSO formargarinehardstock
18:2linoleic
18:0stearic
18:1oleic∆9
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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
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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
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Availability of high-oleic oilsCommercially
availableTechnically
availableOleic(%)Type
cottonseed GM 78 ?
safflower Non-GM 81
sunflower Non-GM 75,85
peanut Non-GM 76
canola Non-GM 60-70
canola GM 88 ?
soybean GM 84 ?
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Adding genes to plantsOilseeds containing long
chain ω3 fatty acids
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Health benefits of ω3 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
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Long chain ω6(n-6) and ω3(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
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Potential new sources of ω3 oils
Marine sources of ω3 fatty acids are declining – will not meet future global needsDietary sources can be expanded by transferring ω3 biosynthetic pathway to plants
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Alternative ω3-PUFA pathways
DHAEPAelongase
∆5 desaturase
elongase
∆4 desaturase
algae, mosses, fungi, nematodes
algae, mosses, fungi, nematodes
polyketide synthesisbacteria, thraustochytrids (anaerobic)
18:4∆6 desaturase
Echium
acetylCoA
18:3
DHAEPA18:4elongase (x2)
∆6 desaturase
β-oxidation
mammals
18:3 ∆6 desaturase
elongase
∆5 desaturase
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Engineering ω3-PUFA in plants16:0
palmitic
18:2linoleic
18:1oleic
18:3linolenic
18:0stearic
∆6∆1518:4SDA
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
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Engineering ω3-PUFA in oilseeds
EPA20:5
SDA18:4
20:4
18:3
DHA22:6
22:5∆5 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
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Engineering ω3-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
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Engineering ω3-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
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Other nutritional improvements
THE FUTURE OF FOOD AND NUTRITION WITH BIOTECHNOLOGY
Removing food allergens through silencing specific proteins • P34 in soybeans
Enhanced β-carotene synthesis to overcome Vitamin A deficiency • Golden Rice, canola oil
Elevated levels of antioxidants• increased levels of α-tocopherol
Increased mineral content • iron in rice from Phaseolis ferretin
protein
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The market will decide
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Adding genes to plantsRenewable sources of
industrial raw materials
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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
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Natural diversity for chemicals
genes fromwild plants
crops
renewableindustrial raw
materials
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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)
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Lauric canola
zero laurate laurate thioesterase genefrom California bay tree
laurate acyltransferasegene from coconut
40% laurate
70% laurate
TE
LPAAT
canola oil
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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
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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?
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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
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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 reductaseβ-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
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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
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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
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Silencing gene expression
Intron
Splicing
Gene
mRNA
Protein
TP
Intron Sequence-specificRNA
Degradation
mRNA Degradation
Double-stranded RNA
No Protein
TP
Coding Region
Enzyme
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Silencing gene expression
Intron
Splicing
Gene
mRNA
Protein
TP
Intron Sequence-specificRNA
Degradation
mRNA Degradation
Double-stranded RNA
No Protein
TP
Coding Region
Enzyme
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Silencing gene expression
Intron
Splicing
Gene
mRNA
Protein
TP
Intron Sequence-specificRNA
Degradation
mRNA Degradation
Double-stranded RNA
No Protein
TP
Coding Region
Enzyme
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Fatty acids are controlled by genesContent of unsaturated fatty acids is determined primarily by three fatty acid desaturase enzymes
16:0
18:1 18:2 18:3
∆9desaturase
∆12desaturase
∆15desaturase
… and the genes that encode them
18:0
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Mid-oleic rapeseed oil (canola)
Selecting naturally-occurring variants
elongasevariants
18:116:0 18:318:2 20:1 22:118:0
354 717 9 262HEAR (rapeseed)
584 926 1 -2LEAR (canola)
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High-oleic canola oil
Selecting naturally-occurring variants
∆12 & ∆15-desaturasevariants
18:116:0 18:318:2 20:1 22:118:0
584 926 1 -2LEAR (canola)
HO-canola (Monola™) 704 320 1 -2
354 717 9 262HEAR (rapeseed)
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Cloned ω3-PUFA synthesis genes
∆5desaturase
H. sapiens
C. elegans
P. patens
Borage
C. purpureus
∆6 desaturase
E. gracilis
∆8 desaturase
C. elegans
n-3 desaturase
H. sapiens
C. elegans
M. alpina
∆5 elongase
∆6 elongase
EPA pks
S. putrefaciens
DHA pks
? C. elegans SchizochytriumM. marinusM. alpina
P. patens
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PUFA biosynthesis in C. elegans∆15 (Fat1)
18:0 18:1 18:2 18:3
18:3
∆6 (Fat3)
18:4
∆6 (Fat3)
GLA SDA
20:4
20:3∆5 (Fat4)
elo1 elo2
∆15 (Fat1)
∆15 (Fat1)
20:4
elo1 elo2
20:5
∆5 (Fat4)
AAEPA