Cellulosic ethanol: huge potential but challenging

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Cellulosic ethanol huge potential but
challenging

• James Dale
• Centre for Tropical Crops and Biocommodities
• Queensland University of Technology

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Alternatives to Fossil Fuels

• Why bioethanol
• Rising oil/fuel prices
• Uncertainty of oil supply political instability
• Peak oil
• Greenhouse gases and climate change

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Relative production costs of alternate fuels
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Plant based ethanol production

• Sucrose is the most attractive feedstock but
  limited supply in Australia
• Grain (starch) is less attractive limited
  supply drain on food/feed supply requires
  enzymatic pre-treatment
• Cellulose biomass extremely promising virtually
  unlimited supply does require enzymatic
  pretreatment.

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Plant based ethanol production

• Sugarcane (sucrose) yeast ethanol
• Sugarbeet (sucrose)

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Sugar
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Plant based ethanol production

• Sugarcane (sucrose) yeast ethanol
• Sugarbeet (sucrose)
• Grains (starch) glucose yeast ethanol
• amylase

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Plant based ethanol production

• Sugarcane (sucrose) yeast ethanol
• Sugarbeet (sucrose)
• Grains (starch) glucose yeast ethanol
• Biomass glucose yeast ethanol
• (cellulose)
• cellulases

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Cellulosic Ethanol Production Process
Cellulases Hydrolysis
Pretreatment
Cellulosic Biomass
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Greenhouse gas savings with biofuels
The percentage greenhouse gas emissions displaced
by driving a unit distance using biofuel instead
of conventional fuel.
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Plant based ethanol production

• Sucrose is the most attractive feedstock but
  limited supply in Australia
• Grain (starch) is less attractive limited
  supply drain on food/feed supply requires
  enzymatic pre-treatment
• Cellulose biomass extremely promising virtually
  unlimited supply does require enzymatic
  pretreatment.
• Where is the opportunity for Australia?

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Sugarcane Biotechnology

• Sugarcane
• A broad-acre tropical crop
• Produces very large biomass per hectare
• gt 200tonnes
• Already an industrial crop harvested and
  processed on a very large scale at centralised
  locations (sugar mills)

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Sugar industry potential - biomass resource

• Production of biomass residues in Queensland
  (tonnes as delivered)
• Cotton trash 17,700
• Macadamia nut shells 10,500
• Cattle dung 70,000
• Fruit processing 4,500
• Municipal waste 695,000
• Timber residues (forest saw mill) 4,164,800
• Total from sources other than sugar industry
  4,962,500
• Bagasse production 10,783,450
• Cane harvest residue (80 of available)
  8,626,760
• Total from sugar industry 19,410,210

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The sugarcane industry current outputs
Conventional Sugarcane
Sucrose
Bagasse
Molasses
Food
Energy (electricity)
Ethanol
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Sugarcane Biotechnology

• Sugarcane
• The future as a biofactory
• Vegetatively propagated (transgene flow)
• Transformation available
• Biolistics
• Agrobacterium
• Field trials of high sugar and disease resistant
  sugarcane in Australia
• Developing the concept of Total Biomass
  Utilisation

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Tropical Crop Biotechnology

• GM Sugarcane

Modified Trait
GM Sugarcane
Sucrose
Bagasse
Biomaterials
Modified Sugars
Food
Cellulose
Lignin
Ethanol
Novel Products
Pulp Products
Ethanol
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Cellulosic ethanol

• Sugarcane is an excellent (renewable) biofuels
  crop
• Cellulosic ethanol is predicted to be the major
  ethanol feedstock (except for Brazil)
• The cellulose degrading enzymes (cellulases)
  necessary to convert cellulose to glucose are a
  very significant component of the cost of
  production unless

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Cellulosic ethanol

• Sugarcane is an excellent (renewable) biofuels
  crop
• Cellulosic ethanol is predicted to be the major
  ethanol feedstock (except for Brazil)
• The cellulose degrading enzymes (cellulases)
  necessary to convert cellulose to glucose are a
  very significant component of the cost of
  production unless

The sugarcane plant produced the enzymes (a free
hit)
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Sugarcane producing cellulases the challenges

• Plant cell walls are made of cellulose
  structural
• Need absolute control over expression of
  cellulases
• No activity during the growth phase normal
  accumulation of sucrose
• High level of expression of cellulases at or near
  harvest

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In planta production of cellulases

• Switch on enzyme synthesis at harvest
• Hydrolyse the cellulose from the inside out
• Much more efficient hydrolysis
• Enzymes provided by the plant no cost
• Requires absolute control over gene expression
• Farmacules INPACT technology transgene
  activation and amplification total control over
  time and place of expression enzymes produced at
  very high levels

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IN
Plant
INPACT
ACTivation

• A two gene activation and amplification
  technology
• A platform technology that provides exquisite
  control over transgene expression activation
  plus amplification
• Is based on the rolling circle replication
  mechanism of ssDNA viruses
• Wide application in agricultural biotechnology
• Specific application in molecular farming
• The technology is being developed to express
  novel molecules in tobacco and sugarcane

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INPACT Technology
Promoter 1
IR
IR
Exon 1
Exon 2
Intron
Intron
Ter
Host
Host
Essential Features

• the viral intergenic region is embedded in an
  intron
• the gene is split into two exons with the second
  exon out of frame - i.e. no product from leaky
  expression
• the split gene is not expressed

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In Plant Activation
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Rep Expression Vector
ethanol
Specific promoter
nos terminator
Alc receptor
nos terminator
Rep/RepA gene
AlcA promoter
Alcohol-inducible Rep/RepA expression
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INPACT Technology
Combinatorial control of gene expression
CaMV 35S
IR
IR
Intron
Intron
Ter
Host
Host
GUS
GUS
and
Rep gene
EtOH induced
Host
Ter
Host
Promoter 1 constitutive CaMV 35S
promoter Promoter 2 inducible ethanol inducible
promoter
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- INPACT Replicating episomes (Replisomes)
Line 2-2
Line 2-3
DAY0
DAY3
DAY3
DAY0
InPAct replisomes
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- Expression levels
GUS stained leaves of a tobacco plant containing
a GUS-based INPACT cassette and alcohol-inducible
Rep/RepA genes, pre and 3 days post ethanol
treatment
EtOH
- EtOH
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Expression levels
60000
50000
40000
constitutive

• ethanol

ethanol
GUS activity (pmol MU/min/mg soluble protein)
30000
20000
10000
0
Alc-GS Alc-GSL (n14)
pINPACT-GUS Alc-REP (n45)
Alc-GS Alc-GSL (n14)
pINPACT-GUS Alc-REP (n45)
35S-GS (n14)
35S-GSL (n14)
pINPACT-GUS (n2)
pINPACT-GUS (n2)
35S 35S promoter Alc Alc promoter GS GUS
syntron GSL GUS syntron LIR n number of
independent transgenic lines
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Western antibody detection of novel protein
(human vitronectin) in tobacco plants transformed
with split gene and Alc-inducible Rep/RepA post
ethanol treatment
ve highest novel protein expressing tobacco
line (line 6) stably transformed with
conventional construct (proprietary promoter
driving gene) 0, 3, 6 DAYS post EtOH treatment
WT 22 12 30
19 10 9 23
ve 0 3 6 0
3 6 0 3 6 0 3 6
ve 0 3 6 0 3 6
0 3 6 0 3 6
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In planta production of cellulases current status

• INPACT adapted to sugarcane
• Developed promoters to completely control
  activation
• Production of toxic/inhibitory proteins in plants
• Optimising cellulase activation and amplification
  in tobacco
• Optimising cellulose hydrolysis using in planta
  produced cellulases
• Optimising sugarcane genetic modification

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Cellulosic ethanol via INPACT
In planta production of cellulases

• Generate GM sugarcane with cellulase genes under
  INPACT control and activator under control of
  inducible promoter
• Grow crop to maturity
• Switch on cellulases 2-3 days pre-harvest by
  spraying with chemical inducer
• Extract sucrose
• Incubate bagasse for glucose production
• Fermentation and distillation

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Cellulosic Ethanol Production Process
Cellulases Hydrolysis
Pretreatment
Cellulosic Biomass
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In Planta Cellulosic Ethanol Production Process
GM Cellulosic Biomass
Incubation
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Acknowledgements
Ben Dugdale Mark Harrison Rob Harding Maiko
Kato Doug Becker Aurelie Chanson Steve
Pirlo Funding Farmacule Bioindustries Australian
Research Council Queensland Government
Mw