The Sugarcane Biofactory
for a sustainable
future
 Why
biomaterials?
Sucrose
Why sugarcane?
 Which
targets?
Sugars
Biopolymers
 Hurdles
 Actions
Isomaltulose
Ethanol
Sorona
Human Population (UN data)
Human
Population
(UN data)Individual Energy Use
Atmospheric
CO
9
8
7
6
5
4
3
2
1
0
9
8
7
6
5
4
3
2
1
0
Year

2
TotalWe are all here
Developed Countries
Most want to be here
00
20
00
18
00
16
00
14
00
Most people in the
world are here
12
00
10
00
20
00
18
00
16
00
14
00
Total
Developed Countries
Billion
We are all here
12
10
00
Billion
Challenges to Sustainability
Year
Exponential increase in (non-renewable) resource consumption
Our World Today
Global Impacts
 Huge
imbalances in resource consumption
 Limited agricultural area - global loss of biodiversity
Ecological
Footprint
Allowing 12 percent for biodiversity, only 1.7 hectares of
biologically productive area per capita is available for human use.
Our World
Today
Global Impacts

Resource depletion
 uneven consumption

Environmental impacts
 loss of biodiversity
 climate change

Global
communications
 rich or poor

Global conflicts
 over resources

Abundant
 ‘guns, germs & steel’
Global
Challenge
Achieve
sustainability
The greatest scientific challenge
in human history
 Renewable bioenergy
& biomaterials
 Plant biotechnology
is a key

 Sugarcane is one of the
most promising crops
Industry Response
(e.g. DuPont):
* 10% of research budget into
renewables from CHO ($130M)
* 25% of sales in renewables
by 2010 ($8B revenues by 2015)
* 100% of vehicle fleet using
renewable fuels by 2015
* Major international biofuels
producer (partner BP)
* Major international bio-based
materials producer (partner MIT)
Harvesting Sunlight
Photosynthesis?
At an energy transformation efficiency of 2%, solar energy collectors covering
1% of land surface with would provide the equivalent of world current oil usage.
?
OR BIOMATERIALS!
Photosynthesis:
Harvesting Sunlight
Why Sugarcane?
Advantages for sustainable biomaterials
High biomass production
» 40-80 tons DW / ha / yr
Simple extraction
» Soluble sugars (20 tons sucrose ≈ 10 kL ethanol/ha/yr)
» Fibre provides energy for processing (excess)
Established gene transfer system
» Into elite cultivars
Inbuilt containment
» No survival outside cultivation
» No pollination of native plants or other crops
» Extraction removes all genes and proteins
The Opportunity
Economic and environmental sustainability

A profitable future based on:
Value-added biomaterials &
bioenergy
 from renewable resources,
 in sustainable & efficient
production systems

Competitive edge from IP
ownership
 built on collaborative R&D

Delivering benefits valued by
customers & consumers
 health & quality of life
 environment
30
IP
25
Value

The Sugarcane Renewable
Biomaterials Industry
20
Biopolymers
15
Valueadded sugars
10
Power & Fuel
5
Sucrose
0
2005
2010
2020
2050
Requirements
for economic viability
The Sugarcane Renewable
Biomaterials Industry
 Platforms
 gene expression patterns
30
 Products
 & market development
Value
 with enhanced value
 Markets
IP
25
20
Biopolymers
15
Valueadded sugars
10
Power & Fuel
5
Sucrose
0
2005
2010
2020
2050
Which Biomaterials?
For sustainable profitability
Biopolymers
Aromatics
Industrial
enzymes
Suit non-food
cultivars
Enhanced
sucrose
yield
Sugarcane
metabolic
engineering
High-value
sugars
Suit food cultivars
Waxes,
pigments,
antioxidants
Biofuel
feedstock
Improved
fibre quality
By-products with
sugar
Which Biomaterials?
For sustainable profitability
Target Compound
Required Production Scale
 Projected world demand (tonnes / year) 2020
 Yield (tonnes / ha) / Area (ha)
Sucrose
Candidate
>130,000,000
20 / 6,500,000
?
<500; low
nil
?
+/+
nil
?
Value
 Price (AU$ / ton wholesale); margin; stability
 Indirect benefits for environment, industry or consumers
Production Method
 Co-production : food / non-food cv? Effect on sucrose yield?
 Alternative to sucrose
Technical Feasibility
 New biological / industrial process needs / likely constraints
 Anticipated capital costs for new industrial facilities
 Research costs and timeline
nil
nil
nil
?
Potential to Capture Value
 Competitors? / Partners?
 FTO / Protected competitive advantage?
 Timing to market
many
+/now
?
Engineering Sucrose Conversion
A pilot study: Isomaltulose
A high-value
sucrose isomer
Isolated genes for sucrose isomerase (SI)
SI
Sucrose ( 1-2 GF)

Isomaltulose ( 1-6 GF)
(= Palatinose)
Benefits from Biofactories
Why Isomaltulose?
Consumer benefits
» Naturally occurring, widely approved
» Non-cariogenic, ‘slow-release’ sugar
» Not fermented by most microbes
» Non-hygroscopic, acid stable
Industry compatibility
» Existing infrastructure
Which plant is more
efficient?
Which is more sustainable?
Downstream potential
» Growing market (potential for value-added blends)
» Precursor for ‘isomalt’

low calorie sweetener
» Potential precursor for petrochemical replacements
Progress with Isomaltulose
Engineered sugarcane
 Express
Storage
parenchyma
an introduced SI gene:
Promoter
NTPP
SI gene
 promoter determines which cells express
 NTPP directs the protein to the vacuole
 SI enzyme converts some sucrose to isomaltulose (IM)
vacuole
SI
cytosol
→
Sucrose ( 1-2)
apoplasm
Isomaltulose ( 1-6)
Some Plants Accumulate IM
Without corresponding decrease in sucrose
SI-expressing Q117 transformants
N3.2
N3.2H
Total sugar concentration (mM)
Q117 control
1000
A
1000
Sucrose
Isomaltulose
Glucose+Fructose
1000
800
800
800
600
600
600
400
400
400
200
200
200
0
0

B
C
0
3 5 7 10 15 20 25 30 35 40 45 50 55 58
3 5 7 10 15 20 25 30 35 40 45 50 55 58
3 5 7 10 15 20 25 30 35 40 45 50 55 58
Internode # from TVD
Internode # from TVD
Internode # from TVD
Results consistent over generations in containment glasshouse
High Total Sugar Content
% Fresh weight
Without corresponding decrease in fibre
85
80
75
70
65
60
30
Water
N3.2H
25
N3.2
Sugar
20
Q117
15
10
Fibre
5
0
0
5
10
15
20
25
30
Internode # from TVD
35
40
High Total Sugar Content
Transgenic sugarcane expressing SI
(mM sucrose equivalents in juice)
Sugar Content
1100
1000
Results in containment glasshouse tests
900
800
700
600
Glucose + Fructose
Sucrose
Isomaltulose
500
400
300
200
100
0
Q117f
Q117d
Controls


UJ2.36
UNJ1.17b
UJC3.7H
UNJ3.2H
SI transformed lines
Some lines accumulate isomaltulose
Some lines show enhanced sucrose accumulation
 implications for biomaterials & bioenergy
 stability and field performance are key considerations
High Total Sugar Content
How does it work?
Sun
light
Storage
parenchyma
Vascular
bundles
CO2
Photosynthesis in
source tissues:
primarily leaf
parenchyma
IM
Sucrose storage
in sink tissues:
primarily mature
stem parenchyma
Futile cycle and
mobilization
Sucrose
transport:
phloem,
symplastic
and
apoplastic
paths
storage vacuole
cytosol
apoplasm
Enhanced Photosynthesis
and sucrose transport
Electron transport
CO2 assimilation
Electron transport rate
(mol e-/m 2/s)
150
100
N3.2H
N3.2
Q117
N3.2H
1200
25
20
15
N3.2H
N3.2
Q117
10
Sucrose uptake (pmol/mg protein)
CO2 assimilation ( Mol CO 2/m2/s)
30
200
Sucrose transport into leaf
plasma membrane vesicles
50
N3.2
1000
Q117
800
600
N3.2+CCCP(at 4')
Q117+CCCP(at 4')
400
N3.2pH8.0
N3.2+CCCP(at 0')
200
0
0
500
1000
1500
2000
Photosynthetically active radiation
(mol photons/m 2/s)
2500
0
500
1000
Photosynthetically active radiation
(mol photons/m 2/s)
0
1
2
3
4
5
Time (min)
6
7
Increased Sink Strength?
Cell wall invertase in storage parenchyma
Central
parenchyma-rich zone
A
**
* **
8
*
CWAI activity
(nmol/gfw/min)
7
*
6
*
9
B
Q117
N3.2
N3.2H
9
8
8
7
7
CWAI activity
(nmol/cm 3/min)
9
Dissected tissues
from central zone
Peripheral
vascular-rich zone
6
5
4
4
3
3
2
2
2
1
1
1
0
3
7
15
Internode # from TVD
20
Parenchyma
*
Vascular
**
6
5
0
C
5
4
3
0
3
7
15
Internode # from TVD
20
Q117
Q117
N3.2
N3.2
N3.2H
N3.2H
Lines
Lines
Reference: Wu L, Birch RG (2007) Doubled sugar content in sugarcane plants modified to produce a
sucrose isomer. Plant Biotechnology Journal 5, 109-117.
SugarBooster Technology
TM
Continuing effort with government & industry
 Working
to establish
 optimal implementation
 stability & efficacy in the field
 genotype specificity
 applicability across species
Potentials
 enhanced sugar accumulation
 enhanced food production
 enhanced biofuel production
 enhanced understanding of
source-sink relationships
IM-producing transformant
Q117 Parent
Total sugar concentration (mM)

1000
1000
800
Glucose + Fructose
Sucrose
Isomaltulose (IM)
800
600
600
400
400
200
200
0
0
3 5 7 10 15 20 25 30 35 40 45 50 55 58
3 5 7 10 15 20 25 30 35 40 45 50 55 58
Internode # from TVD
Internode # from TVD
Progress with other Targets
Sugar derivatives – e.g. Sorbitol
World market ~ 900,000 tons / yr
~ $1,500 / ton
Price
Technology Conversion of cytosolic G-6-P into sorbitol
by apple sorbitol-6-P dehydrogenase
Yields
Leaf: 12% DW
Stem: 1% DW
Challenges
Toxicity – leaf necrosis and stunting
Substrate-limited yield?
Key groups
BSES / CRCSIIB, Australia
Reference
Chong BF, Bonnett GD, Glassop D, O'Shea MG, Brumbley SM (2007)
Growth and metabolism in sugarcane are altered by the creation of a new
hexose-phosphate sink. Plant Biotechnology Journal 5, 240-253.
Progress with other Targets
Polymers – e.g. PolyhydroxyAlkanoates
World market ~ 100,000 tons / yr
~ $1,000 / ton
Price
Technology Conversion of plastid acetyl-coA via 3
bacterial genes into pHB
Yields
Leaf: 2% DW
Stem: <0.01% DW
Challenges
Toxicity in other plants from higher yields
Substrate-limited yield in other plants
Extraction costs (needs >15% DW)
BSES / CRCSIIB, Australia
Key groups
Reference
Petrasovits LA, Purnell MP, Nielsen LK, Brumbley SM (2007) Production of
polyhydroxybutyrate in sugarcane. Plant Biotechnology Journal 5, 162172.
Progress with other Targets
Aromatics – e.g. paraHydroxyBenzoate
World market ~ 10,000 tons / yr
~ $2,400 / ton
Price
Technology Conversion of cytosolic phenylpropanoid
into pHBA by bacterial HCHL enzyme
Yields
Leaf: ~2% DW
Stem: ~1% DW
Challenges
Toxicity in other plants from higher yields
Substrate-limited yield in other plants
Key groups
BSES / CRCSIIB, Australia
Reference
McQualter RB, Chong BF, Meyer K, Van Dyk DE, O'Shea MG, Walton NJ,
Viitanen PV, Brumbley SM (2005) Initial evaluation of sugarcane as a
production platform for p-hydroxybenzoic acid. Plant Biotechnology
Journal 3, 29-41.
Progress with other Targets
Proteins – e.g. Cytokine GM-CSF
Collagen?
World market ~ 200 g @ $1 million / g to
100,000 tons / yr @ $2,000 / ton?
Price
Technology ER-targeted accumulation of constitutively
expressed protein
Yields
0.02 – 1% soluble protein
(~ 1 g / ton cane?)
Challenges
Low yield
Economic extraction
Key groups
USDA – TAMU
HSPA- HARC
Reference
Wang ML, Goldstein C, Su W, Moore PH, Albert HH (2005) Production of
biologically active GM-CSF in sugarcane: a secure biofactory. Transgenic
Research 14, 167-178.
Sugarcane Biofactory
Needs to capture value in Australia
 Platforms
The Sugarcane Renewable
 reliable transgene expression patterns
Biomaterials Industry
 Priority targets
 technical feasibility
 protected competitive advantage
 market appeal
IP
25
Value
 Partnerships - major industry
 market development
 competitive investment level
30
20
Biopolymers
15
Valueadded sugars
10
Power & Fuel
» for delivery & sustainable advantage
 Policy
- government leadership
» corrects historical anomalies
» provides initial markets
» permits industry investment
5
Sucrose
0
2005
2010
2020
2050
Thanks
Visionary support and continuing collaboration
 AusIndustry REDI
 SRDC
 CSR
 ARC – UQ
Collaborations in
platform science


BSES
CSIRO
Leading the teams that do all the hard work





Luguang Wu
Steve Mudge
Mick Graham
Dennis Hamerli
Lianhui Zhang




Terry Morgan
Doug Chamberlain
Jirri Stiller
Annathurai
Gnanasambandam