opium poppy, papaver somniferum alternative products – alternative uses. phil larkin csiro,...
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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.