EPOBIO WORKSHOP 2

GREECE 15-17 MAY 2007Eretria Village Hotel Resort

and Conference Centre

PRODUCTS FROM PLANTS from crops and forests to zero-waste biorefineries

Sugarcane as a

Biofactory

Five genera share common characteristics

The Saccharum ComplexThe Saccharum Complex

1.Saccharum2.Erianthus3.Miscanthus4.Narenga5.Sclerostachya

(Daniels & Roach 1987)

Characterised by:

The Saccharum ComplexThe Saccharum Complex

• High levels of polyploidy • Frequently unbalanced numbers

of chromosomes (aneuploidy)

SugarcaneAverage 35 dry tonnes per hectareHigh Biomass cane >100 dry tonnes per hectare

BSES Limited

Potential Grass Crops

TargetingPlastidMitochondriaPeroxisomeNon-targeted

Subcellular Targetting

Leaf epidermis

Plastid-targeted GFP

Root corticalMature internode

55 amino chloroplast targeting peptide is from the Pisum sativum RUBISCO SSU 3.6 (as in Nawrath et al. 1994 PNAS, 91, 12760-12764) plus a 23 amino acid portion of RUBISCO SSU 3.6, and a 3 amino acid linker.

Anderson et al. Manuscript in press

Green Red

Overlay

Mitochondrial-targeted GFP

Tricome

Anderson et al. Manuscript in press

Epidermis of root cortical cells Immature internode showing pith parenchyma

Mitochondrial-targeted GFP

Vascular bundle from immature internode

Mitochondrial targeting presequence from a Nicotiana plumbaginifolia ATPase β-subunit gene (Boutry & Chua 1985 EMBO J. 4, 2156-2165; Genbank accession X02868)

Anderson et al. Manuscript in press

GFP in the cytosol and nucleusGFP in the peroxisomes

Gnanasambandam et al. Manuscript in prepTillbrook et al. Manuscript in prep

• Vigorous growth

• C4 plant, highly efficient carbon fixation

• Accumulate and store large amounts of carbon as sucrose, plus cellulose and hemicellulose

• Large Biomass

Why Sugarcane?

Production of ρ-hydroxybenzoic acid in Transgenic Sugarcane

Richard B. McQualter, Barrie Fong Chong, KnutMeyer, Drew E. Van Dyk, Michael G. O’Shea, Nicholas J. Walton, Paul V. Viitanen, and Stevens M. Brumbley

McQualter et al. (2005) Plant Biotechnology Journal 3:29-41.

4-coumaroyl-CoA

Chorismate

pHBA

4-hydroxy-benzaldehyde

HCHL

CPL

OGlc

OHO

OH

OGlc

O

OHO CH2

OHO

OHO

OH

SCoAO

OH

HO

OH

OHO

OH

OHH

HCoAS

O

Vacuole

E4P + PEP

Chloroplast

Phenylalanine

Vacuolar uptake

UDP-GT

HCHL

phenolic glucoside and glucose ester*

CPL - chorismate pyruvate-lyaseHCHL - 4-hydroxycinnamoyl-CoA

hydratase/ lyaseE4P - erythrose-4-phosphate PEP - phosphoenolpyruvateUDP-GT - UDP-glucosyltransferase

Cytoplasm

ρ-hydroxybenzoic

Phenylalanine

Ferulic acid

Chlorogenic acid

Caffeoyl-CoACaffeic acid

p-hydroxybenzoic acid

4-hydroxy-benzaldehyde

4-coumaroyl-CoA4-coumaric acidCinnamic acid

Feruloyl-CoA Vanillin Vanillic acid

PAL C4H

+ quinicacid

HQT

4CL

4CL

4CL

COMT

C3H

HCHL

HCHL

NH2

COOH

OH OH

OHCOOH

OHOH

OO

COOH

OH

COOH

OH

COSCoA

OH

CHO

OH

COOH

OH

COOH

OMe

OHOH

COOH

OHOH

COSCoA

OH

COSCoA

OMeOH

CHO

OMeOH

COOH

OMe

CCoAOMT

CCoA3H

PAL - phenylalanine ammonia-lyase

C4H - cinnamate 4-hydroxylase 4CL - 4-coumaroyl-CoA ligaseC3H - 4-coumarate 3-

hydroxylaseCOMT - caffeic acid O-

methyltransferaseHQT - 4-hydroxycinnamoyl-

CoA quinate transferase

Phloroglucinol-staining of lignin in stem sections obtained from TC1 (a) and UH68 (b)

pHBA localization in

leaf & stem tissue that was obtained from UHC1 after 30 weeks growth.

L1

L2L3

L4L5

L6

S5S6

S7

L7

Conclusions pHBA ProjectConclusions pHBA Project• Both CPL and HCHL function in

sugarcane to convert intermediates of existing biochemical pathways to pHBA

• pHBA over-expression demonstrated in leaves and stems of sugarcane

• pHBA expression in leaves highest –7.3% dry wt and increasing!

• pHBA expression in stems – 1.5% dry wt and increasing!

• High correlation between leaf and stem expression

• Preliminary results suggests sugarcane may be an ideal biofactory crop

Sorbitol CaneSorbitol Cane

Malus domestica sorbitol-6-phosphate dehydrogenase gene

Fong Chong, B. et al. Plant Biotechnology Journal 5:240-253.

BSES Limited CSIROBarrie Fong Chong Graham D. BonnettSooknam Patterson Donna GlassopMichael G. O’SheaNial Masel

UQ Chemical EngineeringLars K. NielsenPeter Abedeeya

Sorbitol CaneSorbitol Cane

Malus domestica sorbitol-6-phosphate dehydrogenase (S6PDH) gene

1. S-lines - expressed S6PDH mds6pdh and nptII

2. GS-lines - express S6PDH, glucokinaseand nptII

3. Control lines - nptII

Fong Chong, B. et al. Plant Biotechnology Journal 5:240-253.

Glucose-6-P

Sorbitol

S6PDHSorbitol-6-P

GlucoseFructose

INV

Sucrose

GLK

Fructose-6-P

FK

PGI

SuSy

UDP-glucose

Sucrose-6-P

SPS

Fructose-1,6-BP

FBPase

Glucose-1-P

PGM

UDP-glucose

UGPase

6-phosphogluconate

Ribulose-5-P

Glyceraldehyde-3-P

G6PDH

6PGDH

3-phosphoglycerate

GAPDH

NADPH

NADPH

NADP

NADP

NADPH

NADP

NADPH NADP

Fructose

SDHNADH

NAD

Glucose-6-P

Sorbitol

S6PDHSorbitol-6-P

GlucoseFructose

INV

Sucrose

GLK

Fructose-6-P

FK

PGI

SuSy

UDP-glucose

Sucrose-6-P

SPS

Fructose-1,6-BP

FBPase

Glucose-1-P

PGM

UDP-glucose

UGPase

6-phosphogluconate

Ribulose-5-P

Glyceraldehyde-3-P

G6PDH

6PGDH

3-phosphoglycerate

GAPDH

NADPH

NADPH

NADP

NADP

NADPH

NADP

NADPH NADP

Fructose

SDHNADH

NAD

Sorbitol Cane PathwaySorbitol Cane Pathway

Eight month old S-76 plant (bottom leaf) compared against an equivalent leaf from a control plant (top leaf).

Sorbitol Cane Side EffectsSorbitol Cane Side Effects

Conclusions: Sorbitol CaneConclusions: Sorbitol Cane

Average amount of sorbitol in the leaf lamina –- 120 mg (g dry weight)-1

- 61% of the soluble sugarsin the stalk pith - 10 mg (g dry weight)-1

Sorbitol-producing sugarcane generated 30-40% less aerial biomass and was 10-30% shorter. Leaves developed necroses

pattern characteristic of early senescence severity correlated with amount of sorbitol accumulated.

Production of Polyhydroxyalkanoates

Production of Polyhydroxyalkanoates

• BSES and UQ Chemical Engineering

• ARC Linkage/CRCSIIBMatt PurnellLars PetrasovitsDavid AndersonLihan ZhaoAmy SuKimberley TillbrookPalmina BonaventuraAnnathurai GnanasambandamPeter AbeydeeraLars Nielsen

• Metabolix - AIBN

R groups in PHAsR groups in PHAs

R = hydrogen 3-hydroxypropionate (3HP)

R = methyl 3-hydroxybutyrate (3HB) = PHB

R = ethyl 3-hydroxyvalerate (3HV)

R = propyl 3-hydroxycaproate (3HC)

R = butyl 3-hydroxyheptanoate (3HH)

R = pentyl 3-hydroxyoctanoate (3HO)

R = hexyl 3-hydroxynonanoate (3HN)

• scl-PHA

• mcl-PHA

PHB synthesis in Ralstonia eutrophaPHB synthesis in Ralstonia eutropha

3-ketothiolase

acetoacetyl-CoAreductase

PHB synthase

CH3 O| ||

⎯⎯ O ⎯ CH ⎯ CH2 ⎯ C ⎯⎯ + CoASHn

polyhydroxybutyrate

OH O|

CH3 ⎯ CH ⎯ CH2 ⎯ C ⎯ SCoAR-3-hydroxybutyryl-CoA

||

O||

CH3 ⎯ C ⎯ SCoAacetyl-CoA

O||

CH3 ⎯ C ⎯ SCoAacetyl-CoA

O O

CH3 ⎯ C ⎯ CH2 ⎯ C ⎯ SCoA + CoASHacetoacetyl-CoA

||||

PhaA

PhaC

PhaB

See poster (28) Purnell et al. Spatio-temporal characterisation of polyhydroxybutyrate accumulation in sugarcane

9 month glasshouse study

Sugarcane Leaf

Sugarcane stalk internode

Conclusions: PHB Cane

•Successful transformation of sugarcane with the multigene pathway encoding PHB

•Targeted expression to plastids works well

•Targeting to cytosol, peroxisome and mitochondria not working

•PHB production in sugarcane does not appear to have any negative effects on the transgenic sugarcane plants

•PHB production in sugarcane leaves is continuous over time

•No indication of anything limiting PHB production

Petrasovits, et al. (2007) Plant Biotechnology Journal 5:162-172.

Purnell et al. (2007) Plant Biotechnology Journal 5:173-184.

Summary …Summary …

• 26 PHB positive of 130 sugarcane lines were generated– 6 lines were analysed in a replicated GH trial

• Rank order does not change over time– High and low producers can be detected early

• Accumulation profiles are similar between all lines– PHB accumulates in a time-dependent fashion

• Need for an early detection system

• 26 PHB positive of 130 sugarcane lines were generated– 6 lines were analysed in a replicated GH trial

• Rank order does not change over time– High and low producers can be detected early

• Accumulation profiles are similar between all lines– PHB accumulates in a time-dependent fashion

• Need for an early detection system

Development of an early detection systemDevelopment of an early detection system

• Is Nile Blue A staining at pre-GH stage feasible?– 4 lines passed through tissue culture:

Q117 (0% PHB), B2-12 (0.03%), B3-5 (0.9%), TA4 (2%)

-- 15 plants each were analysed

• Is Nile Blue A staining at pre-GH stage feasible?– 4 lines passed through tissue culture:

Q117 (0% PHB), B2-12 (0.03%), B3-5 (0.9%), TA4 (2%)

-- 15 plants each were analysed

Nile Blue A Screening Randomised Known Samples

Nile Blue A Screening Randomised Known Samples

104100TA456400B3-5011112B2-12000114WT

+++++++/--Line

Negative Weak +ve +ve Strong +ve

This worked with known positives, but what about unknowns?

Nile Blue on unknown samplesNile Blue on unknown samples

7-Jul515176221-Jun1559244534-Jun2595N/S969

screenedDate++++/-Plants

0.05-3.52454545Strong positives0.018-0.098357373Positives0.015-0.02234141Weak positives

0.03110135NegativesRangeHPLCNile blue APlantlets

PositivesNumberCategory300 of these were put in the glasshouse and screened by HPLC

1484 plantlets were screened using Nile Blue A

15 of the 300 lines were selected for further study15 of the 300 lines were selected for further study

PHB content in GH lines at 3 months

00.5

11.5

22.5

33.5

4

L31

L294 L68

L109

L272

L271 L98

L107 L99

L207

L111

L210

L200

L204 L95

L100

Line

% D

W P

HB

as

crot

onic

aci

d

TA4

How are we applying this system?How are we applying this system?

To date, we have generated and screened 5000 plantlets, excluding the 1484 from last year

300+ plantlets are in the glasshouse and >600+ are in tissue culture

These plantlets will be analysed by HPLC at 2 months onward

We have 200 plates of plantlets awaiting Nile Blue screeningEach plate contains ca. 20 plantlets

Currently, we have a further 20 plates of bombarded callus undergoing antibiotic selection and regeneration

We expect ca. 40 plantlets per plate to develop

ConclusionsConclusions

• We have an early detection system for PHB– Results are being written up for publication

• We have developed a high-throughput system for generation of PHB sugarcane– We have processed > 5000 plantlets through

tissue culture and initial screen

• We have an early detection system for PHB– Results are being written up for publication

• We have developed a high-throughput system for generation of PHB sugarcane– We have processed > 5000 plantlets through

tissue culture and initial screen

Petrasovits et al. Manuscript in preparation

MiscanthusSwitch grass

Average yields ~5-6 dry tons/acreUp to 10 dry tons per acre CERES and Noble Foundation

Up to 60 dry tons per hectareUniversity of Illinois at Urbana-

Champaign

Potential Grass Crops

EPOBIO WORKSHOP 2GREECE 15-17 MAY 2007

Eretria Village Hotel Resort and Conference Centre

PRODUCTS FROM PLANTS from crops and forests to zero-waste biorefineries

ThankYou