The Sugarcane Biofactory The Sugarcane Biofactory

Why biomaterials?Why biomaterials?Why sugarcane?Why sugarcane?

Which targets?Which targets?SugarsSugars

Bio-Bio-polymerspolymers

HurdlesHurdles ActionsActions

Sucrose

Sorona

Isomaltulose

Ethanol

for a sustainable for a sustainable future future

Most people in the world are here

Individual Energy Use

Challenges to SustainabilityChallenges to Sustainability

We are all here

Atmospheric CO2

We are all here Most want to be here

Exponential increase in (non-renewable) resource consumptionExponential increase in (non-renewable) resource consumption

Huge imbalances in resource consumptionHuge imbalances in resource consumption Limited agricultural area - global loss of biodiversityLimited agricultural area - global loss of biodiversity

Our World TodayOur World TodayGlobal ImpactsGlobal ImpactsGlobal ImpactsGlobal Impacts

Allowing 12 percent for biodiversity, only 1.7 hectares of biologically productive area per capita is available for human use.

Ecological Footprint

Our World Our World TodayToday

Resource depletionResource depletion uneven consumptionuneven consumption

Environmental impactsEnvironmental impacts loss of biodiversityloss of biodiversity climate changeclimate change

Global Global communicationscommunications rich or poorrich or poor

Global conflicts Global conflicts over resourcesover resources

AbundantAbundant ‘‘guns, germs & steel’guns, germs & steel’

Global ImpactsGlobal ImpactsGlobal ImpactsGlobal Impacts

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)

Global Global ChallengeChallengeAchieve Achieve sustainability sustainability

The greatest scientific challenge The greatest scientific challenge in human historyin human history

Renewable bioenergy Renewable bioenergy & biomaterials& biomaterials

Plant biotechnology Plant biotechnology is a keyis a key Sugarcane is one of the Sugarcane is one of the

most promising cropsmost promising crops

Harvesting SunlightHarvesting Sunlight

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? Photosynthesis?

Photosynthesis: Photosynthesis: Harvesting SunlightHarvesting Sunlight

Why Sugarcane?Why Sugarcane?

High biomass productionHigh biomass production» 40-80 tons DW / ha / yr40-80 tons DW / ha / yr

Simple extractionSimple extraction» Soluble sugars Soluble sugars (20 tons sucrose ≈ 10 kL ethanol/ha/yr)(20 tons sucrose ≈ 10 kL ethanol/ha/yr)

» Fibre provides energy for processing Fibre provides energy for processing (excess)(excess)

Established gene transfer systemEstablished gene transfer system» Into elite cultivarsInto elite cultivars

Inbuilt containmentInbuilt containment» No survival outside cultivationNo survival outside cultivation

» No pollination of native plants or other cropsNo pollination of native plants or other crops

» Extraction removes all genes and proteinsExtraction removes all genes and proteins

Advantages for sustainable biomaterialsAdvantages for sustainable biomaterials

The OpportunityThe OpportunityEconomic and environmental sustainabilityEconomic and environmental sustainability

A profitable future based on:A profitable future based on: Value-added biomaterials & Value-added biomaterials &

bioenergybioenergy from renewable resources, from renewable resources, in sustainable & efficient in sustainable & efficient

production systems production systems

Competitive edge from IP Competitive edge from IP ownershipownership built on collaborative R&Dbuilt on collaborative R&D

Delivering benefits valued by Delivering benefits valued by customers & consumerscustomers & consumers health & quality of lifehealth & quality of life environmentenvironment

SucroseSucrose

Power & FuelPower & Fuel

Value-added sugars

Value-added sugars

Bio-polymers

Bio-polymers

IPIP

The Sugarcane Renewable Biomaterials Industry

RequirementsRequirementsfor economic viabilityfor economic viability

PlatformsPlatforms gene expression patternsgene expression patterns

ProductsProducts with enhanced valuewith enhanced value

MarketsMarkets & market development& market development

SucroseSucrose

Power & FuelPower & Fuel

Value-added sugars

Value-added sugars

Bio-polymers

Bio-polymers

IPIP

The Sugarcane Renewable Biomaterials Industry

Which Biomaterials?Which Biomaterials?For sustainable profitabilityFor sustainable profitability

Sugarcane Sugarcane metabolic metabolic

engineeringengineering

BiopolymersBiopolymers

Industrial Industrial enzymesenzymes

Enhanced sucrose

yield

Waxes, pigments,

antioxidants

Improved fibre quality

High-value sugars

Biofuel feedstock

Aromatics Aromatics

Suit non-food Suit non-food cultivars cultivars

Suit food cultivarsBy-products with

sugar

Which Biomaterials?Which Biomaterials?For sustainable profitabilityFor sustainable profitability

Target Compound Sucrose Candidate

Required Production Scale Projected world demand (tonnes / year) 2020 Yield (tonnes / ha) / Area (ha)

>130,000,00020 / 6,500,000 ?

Value Price (AU$ / ton wholesale); margin; stability Indirect benefits for environment, industry or consumers

<500; lownil ?

Production Method Co-production : food / non-food cv? Effect on sucrose yield? Alternative to sucrose

+ / +nil ?

Technical Feasibility New biological / industrial process needs / likely constraints Anticipated capital costs for new industrial facilities Research costs and timeline

nilnilnil

?

Potential to Capture Value Competitors? / Partners? FTO / Protected competitive advantage? Timing to market

many+ / -now

?

A high-value sucrose isomerA high-value sucrose isomerIsolated genes for sucrose isomerase Isolated genes for sucrose isomerase (SI)(SI)

Sucrose ( 1-2 GF)Isomaltulose ( 1-6 GF)

(= Palatinose)

SI

Engineering Sucrose ConversionEngineering Sucrose ConversionA pilot study: IsomaltuloseA pilot study: Isomaltulose

Which plant is more efficient?

Which is more sustainable?

Benefits from BiofactoriesBenefits from Biofactories

Consumer benefitsConsumer benefits» Naturally occurring, widely approved Naturally occurring, widely approved

» Non-cariogenic, ‘slow-release’ sugarNon-cariogenic, ‘slow-release’ sugar

» Not fermented by most microbesNot fermented by most microbes

» Non-hygroscopic, acid stableNon-hygroscopic, acid stable

Industry compatibilityIndustry compatibility» Existing infrastructureExisting infrastructure

Downstream potentialDownstream potential» Growing market Growing market (potential for value-added blends)(potential for value-added blends)

» Precursor for ‘isomalt’Precursor for ‘isomalt’ low calorie sweetenerlow calorie sweetener

» Potential precursor for petrochemical replacements Potential precursor for petrochemical replacements

Why Isomaltulose?Why Isomaltulose?

Express an introduced SI gene:Express an introduced SI gene:

promoter determines which cells express promoter determines which cells express NTPP directs the protein to the vacuoleNTPP directs the protein to the vacuole SI enzyme converts some sucrose to isomaltulose (IM)SI enzyme converts some sucrose to isomaltulose (IM)

Progress with IsomaltuloseProgress with Isomaltulose

SI geneNTPPPromoter

Sucrose ( 1-2) Isomaltulose ( 1-6)

SI

→

Storage parenchyma

cytosol

vacuole

apoplasm

Engineered sugarcane

Some Plants Accumulate IMSome Plants Accumulate IMWithout corresponding decrease in sucrose

Q117 controlSI-expressing Q117 transformants

N3.2 N3.2H

Results consistent over generations in containment glasshouseResults consistent over generations in containment glasshouse

High Total Sugar ContentHigh Total Sugar ContentWithout corresponding decrease in fibre

Q117

N3.2

N3.2H

Water

Sugar

Fibre

Some lines accumulate isomaltuloseSome lines accumulate isomaltulose Some lines show enhanced sucrose accumulationSome lines show enhanced sucrose accumulation

implications for biomaterials & bioenergyimplications for biomaterials & bioenergy stability and field performance are key considerationsstability and field performance are key considerations

Transgenic sugarcane expressing Transgenic sugarcane expressing SISIS

ug

ar C

on

ten

t (m

M s

uc

rose

eq

uiv

alen

ts i

n j

uic

e)

Controls SI transformed lines

High Total Sugar ContentHigh Total Sugar Content

Results in containment glasshouse tests

High Total Sugar ContentHigh Total Sugar ContentHow does it work?How does it work?

cytosol

storage vacuole

apoplasm

Vascular bundles

Storage parenchyma

Sun light

Photosynthesis in source tissues:primarily leaf parenchyma

Sucrose storage in sink tissues:

primarily mature stem parenchyma

Sucrose transport:phloem, symplasticand apoplasticpaths

CO2

Futile cycle and mobilization

IM

Sun lightSun light

Photosynthesis in source tissues:primarily leaf parenchyma

Sucrose storage in sink tissues:

primarily mature stem parenchyma

Sucrose transport:phloem, symplasticand apoplasticpaths

CO2

Futile cycle and mobilization

IM

Enhanced PhotosynthesisEnhanced Photosynthesisand sucrose transport

Electron transport CO2 assimilationSucrose transport into leaf plasma membrane vesicles

Increased Sink Strength?Increased Sink Strength?Cell wall invertase in storage parenchyma

Q117 N3.2 N3.2H

Lines

Central parenchyma-rich zone

Peripheral vascular-rich zone

Dissected tissues from central zone

Reference: Wu L, Birch RG (2007) Doubled sugar content in sugarcane plants modified to produce a Reference: Wu L, Birch RG (2007) Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. sucrose isomer. Plant Biotechnology JournalPlant Biotechnology Journal 5, 109-117. 5, 109-117.

3 5 7 10 15 20 25 30 35 40 45 50 55 580

200

400

600

800

1000

Q117 Parent

Sucrose

Glucose + Fructose

Isomaltulose (IM)

Internode # from TVD

To

tal s

ug

ar c

on

cen

trat

ion

(m

M)

3 5 7 10 15 20 25 30 35 40 45 50 55 580

200

400

600

800

1000

IM-producing transformant

Internode # from TVD

Working to establishWorking to establish optimal implementationoptimal implementation

stability & efficacy in the fieldstability & efficacy in the field

genotype specificitygenotype specificity

applicability across speciesapplicability across species

PotentialsPotentials enhanced sugar accumulationenhanced sugar accumulation

enhanced food productionenhanced food production

enhanced biofuel productionenhanced biofuel production

enhanced understanding of enhanced understanding of source-sink relationshipssource-sink relationships

SugarBooster TechnologySugarBooster TechnologyContinuing effort with government & industry Continuing effort with government & industry

TM

Progress with other TargetsProgress with other TargetsSugar derivatives – e.g. Sorbitol

World marketPrice

~ 900,000 tons / yr~ $1,500 / ton

Technology Conversion of cytosolic G-6-P into sorbitol by apple sorbitol-6-P dehydrogenase

Yields Leaf: 12% DWStem: 1% DW

Challenges Toxicity – leaf necrosis and stuntingSubstrate-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 TargetsProgress with other TargetsPolymers – e.g. PolyhydroxyAlkanoates

World marketPrice

~ 100,000 tons / yr~ $1,000 / ton

Technology Conversion of plastid acetyl-coA via 3 bacterial genes into pHB

Yields Leaf: 2% DWStem: <0.01% DW

Challenges Toxicity in other plants from higher yields Substrate-limited yield in other plantsExtraction costs (needs >15% DW)

Key groups BSES / CRCSIIB, Australia

Reference Petrasovits LA, Purnell MP, Nielsen LK, Brumbley SM (2007) Production of polyhydroxybutyrate in sugarcane. Plant Biotechnology Journal 5, 162-172.

Progress with other TargetsProgress with other TargetsAromatics – e.g. paraHydroxyBenzoate

World marketPrice

~ 10,000 tons / yr~ $2,400 / ton

Technology Conversion of cytosolic phenylpropanoid into pHBA by bacterial HCHL enzyme

Yields Leaf: ~2% DWStem: ~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 TargetsProgress with other TargetsProteins – e.g. Cytokine GM-CSF

Collagen?World marketPrice

~ 200 g @ $1 million / g to 100,000 tons / yr @ $2,000 / ton?

Technology ER-targeted accumulation of constitutively expressed protein

Yields 0.02 – 1% soluble protein (~ 1 g / ton cane?)

Challenges Low yieldEconomic extraction

Key groups USDA – TAMUHSPA- 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 BiofactorySugarcane Biofactory

PlatformsPlatforms reliable transgene expression patterns reliable transgene expression patterns

Priority targetsPriority targets technical feasibilitytechnical feasibility protected competitive advantageprotected competitive advantage market appealmarket appeal

Partnerships -Partnerships - major industry major industry market developmentmarket development competitive investment levelcompetitive investment level

» for delivery & sustainable advantagefor delivery & sustainable advantage

Policy - Policy - government leadershipgovernment leadership» corrects historical anomaliescorrects historical anomalies

» provides initial marketsprovides initial markets

» permits industry investmentpermits industry investment

Needs to capture value in AustraliaNeeds to capture value in Australia

SucroseSucrose

Power & FuelPower & Fuel

Value-added sugars

Value-added sugars

Bio-polymers

Bio-polymers

IPIP

The Sugarcane Renewable Biomaterials Industry

SRDCSRDC CSRCSR ARC – UQARC – UQ

Visionary support and continuing collaboration Visionary support and continuing collaboration

Collaborations inCollaborations inplatform scienceplatform science

Luguang WuLuguang Wu Steve MudgeSteve Mudge Mick GrahamMick Graham Dennis HamerliDennis Hamerli Lianhui ZhangLianhui Zhang

ThanksThanks

BSESBSES CSIROCSIRO

Leading the teams that do all the hard workLeading the teams that do all the hard work Terry MorganTerry Morgan Doug ChamberlainDoug Chamberlain Jirri StillerJirri Stiller Annathurai Annathurai

GnanasambandamGnanasambandam

AusIndustry REDIAusIndustry REDI