DISCOvery to products: A next generation pipeline for the ... Bio-Engineering/Plant... · •Website: •Duration of 48 months, 8.6 million euro total cost From DISCOvery to products:

• 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