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 ω3 fatty acids (EPA &DHA)Oilseeds producing industrial raw materials

Silencing genes in plantsHigh-oleic cooking oils

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

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 silencing∆12-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 silencing∆9-desaturase

HS-CSO formargarinehardstock

18:2linoleic

18:0stearic

18:1oleic∆9

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 81

sunflower Non-GM 75,85

peanut Non-GM 76

canola Non-GM 60-70

canola GM 88 ?

soybean GM 84 ?

Adding genes to plantsOilseeds containing long

chain ω3 fatty acids

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

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

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

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

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

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

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

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

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

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 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

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 Sequence-specificRNA

Degradation

mRNA Degradation

Double-stranded RNA

No Protein

TP

Coding Region

Enzyme

Silencing gene expression

Intron

Splicing

Gene

mRNA

Protein

TP

Intron Sequence-specificRNA

Degradation

mRNA Degradation

Double-stranded RNA

No Protein

TP

Coding Region

Enzyme

Silencing gene expression

Intron

Splicing

Gene

mRNA

Protein

TP

Intron Sequence-specificRNA

Degradation

mRNA Degradation

Double-stranded RNA

No Protein

TP

Coding Region

Enzyme

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

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)

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)

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

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