1. Sugarcane
EPOBIO WORKSHOP 2
as a GREECE 15-17 MAY 2007
Eretria Village Hotel Resort
Biofactory and Conference Centre
PRODUCTS FROM PLANTS
from crops and forests to
zero-waste biorefineries
2. The Saccharum Complex
Five genera share common characteristics
1. Saccharum
2. Erianthus
3. Miscanthus
4. Narenga
5. Sclerostachya
(Daniels & Roach 1987)
6. Plastid-targeted GFP
Leaf epidermis Mature internode Root cortical
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
8. Mitochondrial-targeted GFP
Epidermis of root cortical cells Immature internode Vascular bundle from
showing pith parenchyma 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
9. GFP in the peroxisomes GFP in the cytosol and nucleus
Gnanasambandam et al. Manuscript in prep
Tillbrook et al. Manuscript in prep
10. Why Sugarcane?
• Vigorous growth
• C4 plant, highly efficient
carbon fixation
• Accumulate and store large
amounts of carbon as
sucrose, plus cellulose and
hemicellulose
• Large Biomass
11. Production of ρ-hydroxybenzoic
acid in Transgenic Sugarcane
Richard B. McQualter, Barrie Fong Chong, Knut
Meyer, 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.
12. ρ-hydroxybenzoic
E4P + PEP
Chloroplast
O OH
Vacuole
Chorismate O OH
Glc
O O Vacuolar O OH
CPL O CH 2
O OH uptake OH
UDP-GT pHBA
OH OH
O
Phenylalanine
Glc
phenolic glucoside and glucose ester* O H
O SCoA
4-hydroxy- Cytoplasm
benzaldehyde
O
OH H
CoAS OH 4-coumaroyl-CoA
CPL - chorismate pyruvate-lyase H
HCHL - 4-hydroxycinnamoyl-CoA HCHL OH
hydratase/ lyase HCHL
E4P - erythrose-4-phosphate OH
PEP - phosphoenolpyruvate
UDP-GT - UDP-glucosyltransferase
15. L1
L3 L2
pHBA L4
L5
localization inL6
leaf & stem
L7
tissue that was
obtained from
UHC1 after 30
S5
weeks growth.
S6
S7
16. Conclusions 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
17. Sorbitol Cane
Malus domestica sorbitol-6-phosphate
dehydrogenase gene
BSES Limited CSIRO
Barrie Fong Chong Graham D. Bonnett
Sooknam Patterson Donna Glassop
Michael G. O’Shea
Nial Masel
UQ Chemical Engineering
Lars K. Nielsen
Peter Abedeeya
Fong Chong, B. et al. Plant Biotechnology Journal 5:240-253.
18. Sorbitol Cane
Malus domestica sorbitol-6-phosphate
dehydrogenase (S6PDH) gene
1. S-lines - expressed S6PDH mds6pdh and
nptII
2. GS-lines - express S6PDH, glucokinase
and nptII
3. Control lines - nptII
Fong Chong, B. et al. Plant Biotechnology Journal 5:240-253.
20. Sorbitol Cane Side Effects
Eight month old S-76 plant (bottom leaf) compared against an
equivalent leaf from a control plant (top leaf).
21. Conclusions: Sorbitol Cane
Average amount of sorbitol
in the leaf lamina –
- 120 mg (g dry weight)-1
- 61% of the soluble sugars
in 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.
22. Production of
Polyhydroxyalkanoates
• BSES and UQ Chemical Engineering
• ARC Linkage/CRCSIIB
Matt Purnell
Lars Petrasovits
David Anderson
Lihan Zhao
Amy Su
Kimberley Tillbrook
Palmina Bonaventura
Annathurai Gnanasambandam
Peter Abeydeera
Lars Nielsen
• Metabolix - AIBN
23. R groups in PHAs
• scl-PHA
R = hydrogen 3-hydroxypropionate (3HP)
R = methyl 3-hydroxybutyrate (3HB) = PHB
R = ethyl 3-hydroxyvalerate (3HV)
• mcl-PHA
R = propyl 3-hydroxycaproate (3HC)
R = butyl 3-hydroxyheptanoate (3HH)
R = pentyl 3-hydroxyoctanoate (3HO)
R = hexyl 3-hydroxynonanoate (3HN)
24. PHB synthesis in Ralstonia eutropha
O O
|| ||
CH3 ⎯ C ⎯ SCoA CH3 ⎯ C ⎯ SCoA
acetyl-CoA acetyl-CoA
PhaA 3-ketothiolase
O O
|| ||
CH3 ⎯ C ⎯ CH2 ⎯ C ⎯ SCoA + CoASH
acetoacetyl-CoA
acetoacetyl-CoA
PhaB reductase OH O
| ||
CH3 ⎯ CH ⎯ CH2 ⎯ C ⎯ SCoA
R-3-hydroxybutyryl-CoA
PhaC PHB synthase
CH3 O
| ||
⎯⎯ O ⎯ CH ⎯ CH2 ⎯ C ⎯⎯ + CoASH
n
polyhydroxybutyrate
25. 9 month glasshouse study
See poster (28) Purnell et al. Spatio-temporal characterisation of polyhydroxybutyrate accumulation in sugarcane
28. 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.
29. 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
30. Development 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
31. Nile Blue A Screening
Randomised Known Samples
Line - +/- + ++ +++
WT 14 1 0 0 0
B2-12 2 1 11 1 0
B3-5 0 0 4 6 5
TA4 0 0 1 4 10
Negative Weak +ve +ve Strong +ve
This worked with known positives, but what about unknowns?
32. Nile Blue on unknown samples
1484 plantlets were screened using Nile Blue A
Plants +/- + ++ Date
screened
969 N/S 95 25 4-Jun
453 24 59 15 21-Jun
62 17 15 5 7-Jul
300 of these were put in the glasshouse and screened by HPLC
Category Number Positives
Plantlets Nile blue A HPLC Range
Negatives 135 0 1 0.031
Weak positives 41 41 3 0.015-0.022
Positives 73 73 35 0.018-0.098
Strong positives 45 45 45 0.05-3.52
33. 15 of the 300 lines were selected for further study
PHB content in GH lines at 3 months
4
% DW PHB as
crotonic acid
3.5
3
2.5
2 TA4
1.5
1
0.5
0
L31
L294
L68
L109
L272
L271
L98
L107
L99
L207
L111
L210
L200
L204
L95
L100
Line
34. 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 screening
Each 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
35. Conclusions
• 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
36. Potential Grass Crops
Miscanthus
Switch grass
Average yields ~5-6 dry tons/acre
Up to 10 dry tons per acre
CERES and Noble Foundation
Up to 60 dry tons per hectare
University of Illinois at Urbana-
Champaign
37. Thank EPOBIO WORKSHOP 2
GREECE 15-17 MAY 2007
You Eretria Village Hotel Resort
and Conference Centre
PRODUCTS FROM PLANTS
from crops and forests to
zero-waste biorefineries
