Opium poppy, Papaver somniferumAlternative products – alternative uses.

Phil LarkinCSIRO, Canberra

Australian poppy industry

• >50% of world’s legally traded opiates

• Demonstrated the power of genetics to transform the industry to new products– Thebaine poppies (top1 mutation)– Thebaine feedstock for high value painkillers, such as

buprenorphine, oxycodone.

Top1 mutation

CO52 ThebaineControl mutant

Zero morphine and codeine.

Proprietary mutation of Tasmanian Alkaloids

Morphine-pathway block in top1 poppies.Millgate, Pogson, Wilson, Kutchan, Zenk, Gerlach, Fist, Larkin.Nature 431:413-414 (2004)

Transgenic poppies

• Biotechnology is another strategy capable of transforming the industry:– Increasing morphinan content– Novel products

Hypocotyl explants

Agrobacterium

Black primary callusWhite embryogenic

callusSomatic embryos

Transformation of Poppy

Metabolic Engineering of Opium Poppy

Increasing alkaloid yield

thebaine

morphine

analgesics

Engineering novel products

reticuline

morphine

hpRNAi anticancerand antimalarialactivities

0

1

2

3

4

5

6

NTg Tg

#1b

NTg Tg

#2

n=2

n=6

n=1

n=7

CO

R t

ran

sc

rip

t(n

orm

alis

ed a

rbit

rary

un

its

)

Transgenic over-expression of COR (codeinone reductase) increases yield

• COR gene transcript is increased

• Morphinan alkaloid content is increased …..

Control - -

206-

4-1a

206-

4-1b

206-

4-2 -

206-

4-4

206-

4-7

206-

4-8

0.0

0.5

1.0

1.5

2.0

2.5

3.0

29%*** **

**

alka

loid

%D

W

Control 2

Control 3

Control m

ean

206-

4-1a

206-

4-1b

206-

4-2

206-

4-3

206-

4-4

206-

4-7

206-

4-8

0.0

0.5

1.0

1.5

2.0

2.5

3.0

27%

* *

alka

loid

%D

W

Control 2

Control 3

Control m

ean

206-

4-1a

206-

4-1b

206-

4-2

206-

4-3

206-

4-4

206-

4-7

206-

4-8

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

28%* * **

* * *

alka

loid

%D

W

GH2002 GH2003

FIELD2003

Cor transgenics

GH2004

CO58-3

4

206-

4-1b

206-

4-1b

HOMa

206-

4-2

206-

4-2H

OMa

206-

4-2H

OMb

206-

4-2H

OMc

206-

4-3

206-

4-7

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5 3 3 3 3 3 3 3

40%

Alk

alo

id %

DW

COR over-expression – Field TrialField 2003% DW

Morphine Codeine Thebaine Total

CO58-34control

2.83 0.19 0.21 3.25

1a 3.43 * 0.24 0.22 3.91 *

1b 3.32 0.31 * 0.32 3.97 *

2 3.44 * 0.31 * 0.38 4.17 **

3 3.31 0.30 * 0.28 3.95 *

4 3.38 * 0.27 0.26 3.92 *

7 3.45 * 0.25 0.18 3.89 *

8 3.16 0.22 0.19 3.60* , P<0.05**, P<0.01

Control n=18, 20 plants per replicateTransgenics n=6, 20 plants per replicate

Metabolic engineering for alternative products

• Zero opium poppy by genetic engineering

• CSIRO gene silencing technology to shut down all opiates.– Hairpin RNAi – E.g. Target gene: codeinone reductase– Accumulation of alternative non-opiate

product, reticuline.

Opiate-free poppy

codeinone reductase

Opiate-free poppy

Potential feedstock for other bioactives

Allen, RS et al. (2004) RNAi-mediated replacement of morphine with nonnarcotic alkaloid reticuline in opium poppy.Nature Biotechnology 22, 1559-66.

Opiate-free poppy

T

O

C

M

LsLn

CmR

Transgenicpoppies

Control poppiesstandards

Morphine, codeine and thebaine replaced by reticuline, codamine, laudanine and laudanosine.

Proposal for Afghan Poppy Industry

Phased transformation to a legal non-opiate industry:

• Phase 1. Biodiesel from poppy seed oil.• Phase 2. Higher value “soft” biodiesel.• Phase 3. Industrial and pharmaceutical products.

Poppyseed oil for biodiesel.

• Current world diesel consumption 1087 billion litres p.a.• Biodiesel currently accounts for 0.4%.• Poppy yields 1.8 tonnes/ha seed (cf. 2.4 for canola)• 45-50% oil content (cf. 40% in canola)• 0.8 tonnes oil per ha.• Afghan poppy area in 2004 was 131,000 ha

– <2 % of arable land– Would produce 100,000 tonnes of oil – = 2.5% of current world biodiesel consumption.– Scope to expand production greatly.

• Protein “meal” byproduct as food and feed.

Phase 1. Biodiesel from poppy seed oil.

• Replacement seed of opiate-free poppies mutation– Mutant cv. Sujata, opiate-free, opium-free poppy

(CSIR Lucknow, India)• Village-scale oil press and biodiesel processing• Local energy supply

– Transport fuel– Electricity generators

• Basis of a carbon-neutral export biodiesel industry.• Low tech and high volume• Requiring minimal adjustment from the poppy growing culture and

expertise.• Begins the process of legalising the industry:

– Agronomic support– Potential Carbon subsidies– Breeding support– Trade support

Phase 2. Higher value “soft” biodiesel.

Value adding to poppy oil with biotechnology• Increased oil yield

– Overexpressing DAGAT2 (boost transfer of last acyl to glycerol)

• High oleic biodiesel.– Polyunsaturates can form oxidised polymers which reduce engine

performance.

– High oleic oils (monounsaturates) are preferred.

– Well attested CSIRO technology used to produce high oleic oils in soybean, canola, flax and cottonseed oils:

• RNAi silencing 12-desaturase (high oleic)• RNAi silencing FATB thioesterase (eliminate palmitic)

• High oleic poppy oil also a healthier food oil.