• Website: www.disco-fp7.eu• Duration of 48 months, 8.6 million euro total cost
From DISCOvery to products: A next generation pipelinefor the sustainable generation of high-value plant
products
Prof Paul Fraser, Head of Plant Molecular SciencesRoyal Holloway University of LondonCoordinator EU-FP7 DISCO project
The shift from “black” (petrochemical based) to “green” (plant biomass)
• Presently 4,129 mill tonnes oil consumed globally - 97% fossil fuel and 3% renewable.
• By 2025 - 3 to 8% renewable source is required - equalivent to 400 billion litres.
• 2050 - 27% from a renewable sources is required- 100 million hectares compared 30 million presently.
Werpy and Petersen, 2014. PBD; Fresenko and Edwards, 2014 J.Exp.Bot. 65 1927.
Bio-based versus petro-based refinery concepts
DISCO Aims and objectives
Aim: Pipeline from Discovery to the Production of Sustainable Bioproducts
Objectives
1. Bioprospecting: Exploit existing biodiversity collections in Solanaceae and Iridaceae for bioactives using high-throughput bioassays.
2. Pathway elucidation utilising modern transcriptomictechnologies
3. Development of enabling technologies
4. Generation of new biosources of high-value plant derived products
5. Develop down-stream processes with reduced environmental impact.
6. Demonstrate technical and economic production feasibility.
P1: Paul D. Fraser (coordinator), School of Biological Sciences, Royal Holloway University of London, UK. [RHU]P2: Ralph Bock, Max-Planck Institute of Molecular Plant Physiology, Potsdam, Germany. [MPI]P3: Joseph Hirschberg, Department of Genetics, Institute of Life Sciences, Israel. [HUJ]P4: Giovanni Giuliano, Casaccia Research Center, Ente Per Le Nuove Tecnologie, L’Energia E L’Ambiente, Roma, Italy. [ENEA]P5: Wolfgang Schuch, Fraunhofer, Chile. [FCR]P6: Derek Stewart, James Hutton Institute, Invergowrie, Dundee, UK. [SCRI]P7: Oliver Kayser, Dortmund , Germany. [TuDo]P8: Efraim Lewinshon, Volcani Centre , IL [ARO]
DISCO ACADEMIC PARTNERS
P9: Carmen Socaciu, Proplanta SRL, Romania. [PRO]P10: Philippe Thonart, Fermented Product Partner, Belgium. [FPP]P11: Verena Peuser, European Research Office, Germany.[EURICE]P12: Fabien Havas, Israeli Biotechnology Research Ltd., Isreal. [IBR]P13: Ian McPhee, Scottish Biomedical, UK. [SBM]P14: Federica Cattonaro, IGA technology Services, Italy. [IGA]P15: Hansjorg Hagels, Boehringer , Germany. [BOE]
DISCO INDUSTRIAL PARTNERS
Module 3TRANSLATION
Module 2 APPLICATION
Module 1 DISCOVERY
WP2
Bioprospecting
WP3
Bioactivity
WP3
Pathway discovery
WP5
Enabling technologies
WP6
Genetic intervention
WP7
Down stream processing
WP8
Production feasibility
WP9
Product effectiveness
P1, P4, P6, P7, P8
P13 &P14
P1, P2, P3, P4, P6, P7, P9, P10, &
P12
P1, P5, P9, P10, P12, P15
Interaction and workflow of DISCO activitiesApoCar
CAR SPSOLApoCar
SP SOL SOL
WP 1-Management, WP 10-Training & WP11-DisseminationP11
EURICE
Plant transformation
• Either Agrobacterium mediated
• Direct particle bombardment
The Plastid
Nucleus
Vacuole
E2E1
P1 P2 EP
+TE112
3
4 TE1TE1
Mini-pathways
5
5E1 E2 HE
E1 E2 HE
5
7
Intermediary
metabolism
7CP
Sequestration
9
9
Modification
6Plastid transformation
HE
NPs
8 New
Products
Degradation
14
Wild type Wild type Wild typeVariety-1 Variety-2 Variety-3
10. Increased plastids 11. Larger plastids 12. Different plastid specialisation
TFs
Approaches to the metabolic Engineering of natural product pathways in plants
Approaches to the metabolic Engineering of natural product pathways in plants
Host platforms: Solanaceae
• Sequenced genomes• Vast exploitable natural variation• Amenable to genetic intervention• Can be contained• Biochemical platforms established• Possess sink organs• Rich in secondary metabolites
Target: The alkaloid pathway- Scopolamine
• Pharmaceutical use• Present sources Solanaceae spp. Duboisa and Datura
Target bioactives: Carotenoids/apocarotenoid pathway
PhytoeneColourless Carotenes: Phytoene
Cosmetic industry
Saffron derived apocarotenoids
Food sector
KetocarotenoidsFood/feed and Health
sector
Target: Terpenoid pathway solanesol
SolanesolHealth and pharma sectors
• Present in Solanaceae species• Present sources are unreliable e.g. tobacco waste• Susceptible to supply and price fluctuations• Used in the synthesis of coenzyme Q9 • Anticancer treatments
Jones, MO., Perez-Fons, L., Robertson, FP., Bramley, PM. and Fraser PD. (2012). Functional characterisation of long chain prenyl diphosphate synthases from tomato. Biochem. J., doi:10.1042/BJ20120988. 10.1093/jxb/ers252.
MVA
GPP
FPP Squalene
Amyrins
Saponins
Phytosterols
Acetyl CoA
DMAPP
Apocarotenoids
Monoterpenes
GGPP
IPP DMAPP
FPP
GPP
Pyr + G-3-PO4
DXP
Diterpenes
Chlorophyll
Tocopherols
Carotenoids
Xanthophylls
Absiscic acid
Isoprene
Cytokinins
Tocotrienols SPP
Plastoquinones
Phylloquinones
PLASTID
IPP
GGPP
Monoterpenes
Polyterpenes
Geranylgeranyl proteins
Gibberellins
Ubiquinone IPP
MITOCHONDRIA
CYTOSOL
Isoprenoid Biosynthesis
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3 CH3
CH3
CH3
CH3
CH3
CH3 CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3 CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3 CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3 CH3 CH3 CH3
CH3 CH3CH3CH3
CH3
CH3
CH3
CH3
CH3
CH3CH3
CH3
CH3
CH3
CH3
CH3 CH3
CH3 CH3CH3CH3
CH3CH3
CH3
CH3 CH3
CH3 CH3
CH3CH3
CH3
CH3 CH3
CH3
CH3 CH3
CH3 CH3
CH3CH3
CH3
CH3 CH3
CH3
OH
OH
CH3 CH3
CH3 CH3
CH3 CH3
CH3OH
OCH3CH3
CH3OH
O
CH3 CH3
CH3 CH3
CH3 CH3
CH3OH
CH3CH3
CH3OH
O
CH3
CH3 CH3
CH3 CH3CH3CH3
CH3CH3
CH3
CH3 CH3
CH3 CH3
CH3CH3
CH3
CH3 CH3
CH3
CH3 CH3
CH3 CH3
CH3CH3
CH3
CH3 CH3
CH3
OH
OH
CH3
CH3CH3 CH3 CH3
CH3
CH3CH3
CH3OH
OHCH3
CH3 CH3
CH3
CH3CH3
OH
O
CH3
OPP
.
15-cis-Phytoene
15,9′-dicis-Phytofluene
9,15,9′-tricis-z-carotene
9, 9′-dicis-z-carotene
All-trans-lycopene7,9,7′9′-tetracis-lycopene
7,9,9′-tricis-Neurosporene
d -carotene
a -carotene
g-carotene
b-carotene
lutein
GGPP x2
zeaxanthin
9-cis-neoxanthin
violaxanthin
antheraxanthin
IPP
PSY-1
PSY-2
PDS
PDS
light
ZDS
ZDS
CRTISO
LCY-E
LCY-B
LCY-B
LCY-BCYC-B
CYC-B
CRTR-B1
CYP79A
CRTR-B1
CYP97A
CYP97C
NXS
VDE-1
VDE-1 ZEP-1
ZEP-1
light
CRTBR-B2
Carotenoid Biosynthesisin higher plants
Why enhance levels in plants?
• Improve nutritional properties of foodstuffs.
• Confer health benefits via dietary intake
(e.g. lycopene, zeaxanthin and b-carotene).
• Most isoprenoids have annual markets over $ 1 billion.
• Replace chemical synthesis to improve environmental impact.
• Overcome low-yielding, slow growing plant sources not amenable to agricultural production.
High Lycopene
High
Provitamin A
Canola
“Golden Rice”
Production of Golden rice an humanitarian GM product
• 4 million lives lost per year in Sub-Saharan Africaand South-East Asia
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
b -carotene
(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Daffodil gene
Single bacterial gene;
performs both functions
Daffodil gene
The absence of the b-Carotene biosynthetic pathwayin rice endosperm
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
b -carotene
(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Rice lacks
these enzymes
Com
ple
teV
itam
inA
Path
way
Carotenoid enhancement in rice by introduction of psyorthologues and crtl
HPLC-PDA analysis of the carotenoid extracts
A. Control endosperm, B to D are different events showing difference carotenoid profiles
Golden Rice II – production has been shown to be technically feasible andits effectiveness demonstrated with human intervention trials
cis-Phytofluene
GGPP
15-cis-Phytoene
cis-z-Carotene
cis-Neurosporene
Poly-cis-Lycopene
All-trans-Lycopene
b-Carotene
Zeaxanthin
Violaxanthin
Antheraxanthin
Neoxanthin
d-Carotene
a -Carotene
Lutein
Zeinoxanthin
g-Carotene
PSY-1PSY-2
PDS
PDS
ZDS
ZDS
CRTISO
CYC-B CYC-B
LCY-B
LCY-B LCY-B
LCY-E
CRTR-B1 CRTR-B2
CRTR-B1/2
CRTR-E
NXSZEP
ZEP VDE
VDE
CHL+
Light
Vegetative tissue
Ripe fruit tissue
Non-enzymatic
Both vegetative and
ripe fruit tissue
Light
Predominant in
vegetative tissue
Predominant in
ripe fruit tissue
Carotenoid Biosynthesis in tomato
fruit chloroplasts and chromoplasts
ControlDXS
HMG
R IPICrtE
CrtBCrtILCY
DXS+CrtE
DXS+CrtB
DXS+CrtI
CrtE+CrtB
CrtE+CrtI
CrtB+CrtI
PSY-1+CrtB
0
1
2
3
4
Rel
ativ
e fo
ld i
ncr
ease
ControlDXS
HMG
R IPI
CrtECrtBCrtI
LCY
DXS+CrtE
DXS+CrtB
DXS+CrtI
CrtE+CrtB
CrtE+CrtI
CrtB+CrtI
PSY-1+CrtB
0.0
2.5
5.0
7.5
Rel
ativ
e fo
ld i
ncr
ease
IPP
HMG-CoA
HMGR
IPP DMAPP
Pyr + G-3-P
DXP
GGPP
Phytoene
Lycopene
b-Carotene
PLASTID
DXS
CrtE
CrtB
CrtI
LCY
b-Carotene
Lycopene
Pathway Engineering: Transgenic varieties with single/multiple Steps in the pathway amplified
IPI
0.1
0.1
0.1
0.4
Flux control coefficients
C Ji/Ei
Rainbow trout
Atlantic salmon
Chicken
Carotenoids as feed supplements
Aquaculture
Poultry feed
Biotechnological features of the ketocarotenoids
• Used in food and feedstuffs as a natural colorant
• Global markets $1 billion per annum
• The necessity to supply ketocarotenoids in aquaculture feed accounts for 25% of the total aquaculture costs
• Chemical synthesis present method of choice, complex and expensive
• No suitable plant based renewable sources exist
4, 4’-b-oxygenase (ketolase) reaction
• Several micro-organisms Brevundimonas spp, Paracoccus spp, Haematococcus spp, contain an enzyme capable of introducing keto groups into the 4 position of carotenoid b-rings.
Potential precursors and products formed by the oxygenase/hydroxylase combination
Fraser et al., 1998. Eur. J. Biochem., 252, 229
Endogenous precursorsin plants
Fraser et al., 1997. J. Biol. Chem., 272, 6128
Engineering Ketocarotenoid formtaion into Nicotiana glauca
WT ZW
Astaxanthin
4-ketozeaxanthin
β-carotene
Echineone
Canthaxanthin
Phoenicoxanthin
WT ZW
Ketocarotenoids are1.0 to 3.0% DW
WILD TYPE Nicotiana glauca
TRANSGENIC
Plastoglobule
Plastoglobule
Endogenous Peaks:
1. Lutein
2. β-carotene
3. cis-β-carotene
Unique Peaks:
4. 4-Ketolutein
5. Unknown
6. 3-OH-echineone epoxide
7. Astaxanthin
8. 4-Ketozeaxanthin
9. Phoenicoxanthin
10. 3’OH-Echinenone
11. Canthaxanthin
12. 3-OH Echinenone
13. Echinenone
AU
0.00
0.05
0.10
0.15
Minutes
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
AU
0.00
0.20
0.40
Minutes
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
1 2-3
1 2-3
AU
0.00
0.20
0.40
Minutes
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
4 5
67
8
9
10
11
1213
Ketocarotenoid esters
A: Control
B: Transgenic
Comparative Profiles of carotenoids present in Transgenic Tobacco flowers expressing ketocarotenoid biosynthetic gene products
Transgenic potato expressing astaxanthin biosynthetic genes
B C
E F
A
D
(a) (b)
Plastid
envelope
Lipid
Fraction
Mortimer et al. 2015 Plant Biotechnol. J., in press
Transgenic tomato lines expressing ketocarotenoidbiosynthetic genes
Wild type
Transgenic lines
Minutes
4.20 8.40 12.60 16.80 21.00 25.20 29.40 33.60 37.80 42.00
Minutes
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
Minutes
4.20 8.40 12.60 16.80 21.00 25.20 29.40 33.60 37.80 42.00
Minutes
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
15 30
15 30
minutes
minutes
0.2
0.1
0.2
0.1
AU
AU
Violaxanthin
Neoxanthin
Chlorophyll b
Lutein
Chlorophyll a
Beta-carotene
Astaxanthin
Phoenicoxanthin
Canthaxanthin
4-Keto-lutein
Echinenone
Mono-esters
Di-esters
Wt fruit
Transgenic Z/W plants
HPLC analysis of ketocarotenoid producing tomato fruit
Ultrastructural changes
Summary
• Prototype High lycopene, b-carotene, canthaxanthin,phoenicoxanthin (adonirubin) and astaxanthin3 to 25 mg /gDW, 170 kg per hectare.
• Difficult to separate biosynthesis from accumulation
• New products can undergo unpredicted chemicalmodification.
• Fundamental lack of understanding with regard to the regulation of pathways
Cultivation Scale-up polytunnel facilities
b-Carotene Fruit
KetocarotenoidFruit
Leaf phenotypes
Vegetative material Fruit material
Extraction, enrichment& purification
Aquaculture
Poultry feed
Biogas production
Solanesol
+ Metabolite
profiling
Biomass utilisation
Processing for product evaluation
Summary
• Tomato fruit can be used as a cell factory
• Demonstrated that plant based renewable sources can compete with chemical synthesis
AcknowledgementsRHUL
Marilise NogueiraGenny EnfissiLaura Perez
Fatemeh KermaniLeticia MoraHarriet Berry
Cara MortimerMargit Drapal
Elliott PriceAmanda Kozlo
Eugenio AlcadeJack Gillan
Rebecca NohlNatalia Carreno
Martin KlopmakerChris GerrishPeter Bramley
DISCO PARTNERS
Nottingham UniversityGraham Seymour
Catrin Rutland
Ishikawa UniversityNorikiho Misawa
AgrobiosGiovanni Giorio
EU FP7-METAPRO project No. 244348EU FP7-MultiBioPro project No. 311804EU FP7-DISCO project No. 613513ERA-CAROMAIZE
Syngenta Ltd.Charles Baxter
Frankfurt UniversityGerhard Sandmann