Biomass Oils: Allan Green Research Director. Game-changing new technology for biofuels and bioproducts. CSIRO Bioproducts Program

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1 Biomass Oils: Game-changing new technology for biofuels and bioproducts. Allan Green Research Director CSIRO Bioproducts Program Thomas Vanhercke James Petrie Ben Leita Surinder Singh

2 Turning over a new leaf Leaf cells are normally like this Rich in starch and other polysaccharides Oil droplet Starch granules but very low in oils (TAG < 1%)

3 Turning over a new leaf Now they can be like this Engineered to accumulate high levels of oil (TAG) Equivalent to seed oil levels (35% + )

4 Game-changing technology platform Greatly expands global capacity for renewable oil production Enables intensification of oil production Lowers plant oil feedstock cost Potentially enables price-competitive biodiesel based fuels

5 Plant oils (triglycerides) Clean, renewable sources of energy and useful carbon skeletons CO 2 H 2 O Nutrients Solar energy Up to 60% triglycerides (seed storage oils) Food uses (80%) Industrial uses (20%) Energy Calorific value (ME) Heat transfer (cooking) Engine power (biodiesel) Molecules Fatty acid nutrition Lubricants Chemical feedstocks

6 The need to increase plant oil production 250 To provide surplus for significant petroleum replacement Industrial Food use 160 MT 250 MT To keep pace with growing food oil needs

7 Oil production trebled in past 25 yrs Doubling area sown to oil crops (oil palm, soybean and rapeseed/canola) Strong gains in oil yield per hectare through 40-50% improvement in crop productivity Improvements in oil content were relatively minor

8 Further acreage expansion is limited Arable land area is under increasing pressure. Rate of expansion will slow down significantly Trebling oilseed acreage would require an additional 200 Mha + 60 Mha + 25 Mha

9 Oilseeds are under-performing Macadamia 70% Sesame 60% Sunflower Rapeseed 48% 48% Peanut 48% Safflower 38% Soybean Cottonseed Lupin 19% 18% 8%

10 Oilseeds are under-performing Macadamia 70% Sesame 60% Sunflower Rapeseed 48% 48% 7% 7% Peanut 48% 7% Safflower 38% 17% Soybean Cottonseed Lupin 19% 18% 8% 36% 37% 47% 55%

11 Oilseeds are under-performing Macadamia 70% Sesame 60% Sunflower Rapeseed 48% 48% Peanut 48% Safflower 38% Soybean 19% 36% X 260 MT + 94 MT Cottonseed 18% 37% X 39 MT + 14 MT Lupin 8% 55% MT

12 How can we increase plant oil production? New plant oil sources 250 Maximising seed oil content Industrial Food use 160 MT 250 MT Productivity gains (1% pa)

13 Push

14 Amplify fatty acid synthesis and plastid export enzymes (using transcription factors) LEC1 LEC2 WRl1 Carbon Sucrose import & cleavage Glycolysis OPPP Late glycolysis Fatty Acid synthesis TAG assembly FA modification TAG storage Redirect carbon flux away from competing pathways (starch, protein)

15 Express acyltransferases (DGAT or PDAT) to convert DAG to TAG +

16 Prevent TAG breakdown by silencing endogenous lipases Lipase SDP1 CSIRO proprietary hprnai gene-silencing technology

17 Encapsulate oil droplets in protective coatings (oleosins or LDAPs) Oilbody protein Phospholipid membrane Triglycerides (oil) Oilbody

18 Transient pathway expression Multiple transgenes simultaneously infiltrated into N. benthamiana leaf Complete DHA pathway assembled and optimised by combinatorial screening DHA oilseeds

19 Transient pathway expression Push & Pull SYNERGY! 2.5% WRI1 DGAT1 TAG (% DW) 0.5% WT WRI DGAT WRI DGAT

20 Stable expression in tobacco High biomass species with stable transformation system wt TG Oil = 20% leaf DW

21 Leaf oil heat map Oil % DW WT LEC2 SDP

22 1% 25% Other Lipid Protein Ash Starch Fibre (TDF) Other CHO 3 % 4 % 7 % 28 % 29 % 28 % Other Lipid Protein Ash Starch Fibre (TDF) Other CHO 2 % 4 % 7 % 24 % 16 % 22 % Whole leaf Oil-free residue

23 1% 25% Other Lipid Protein Ash Starch Fibre (TDF) Other CHO 3 % 4 % 7 % 28 % 29 % 28 % Other Lipid Protein Ash Starch Fibre (TDF) Other CHO 2 % 4 % 7 % 24 % 16 % 22 % Fatty acid (%) 16:0 18:0 18:1 18:2 18:3 48 % Fatty acid (%) 16:0 18:0 18:1 18:2 18:3 6 %

24 Technology refinements more oil OIL (% DW) 40 34% oil seed set) FA (% Oil)

25 Tobacco: potential first leaf oil crop spaced row crop increased plant density high biomass genotype 10 T/HA 15 T/HA 20 + T/HA (Oil yield = oil palm)

26 Dedicated high-biomass oil crops Tobacco leaf

27 Dedicated high-biomass oil crops Sugarcane

28 Dedicated high-biomass oil crops Sorghum

29 Dedicated high-biomass oil crops Alfalfa

30 High-oil crop residues: Corn stover

31 High-oil crop residues: Wheat straw

32 Techno-Economic evaluation BIOMASS COSTS Farm-gate value Oil content Yield trade-off LOGISTICS COSTS Freight rate Catchment radius Inventory costs PROCESS COSTS Oil extraction Biomass conversion Residue value

33 Competitive production cost Cost of Production US$/bbl Corn Corn Cellulosic OPEC Deep water GTL CTL Oil EOR Arctic Oil shale Sugar sands cane Leaf?

34 CSIRO has strong patent position First to demonstrate oil >10% oil in leaves. Substantially higher levels obtained through Push/Pull. Invention is founded on synergistic effects of FAS transcription factors and TAG assembly enzymes Priority date 2011 Granted claims in USA (2014) Extensive family of international filings incorporating additional technology enhancements

35 Game-changing new platform Greatly expands global capacity for renewable oil production, to all plants Leaf oil composition can be engineered for specific purposes (food, nutritional, industrial FA, fuel) Enables intensification of oil production for sustainability Lowers plant oil feedstock cost, potentially enabling price-competitive biodiesel

36 Collaboration opportunities CSIRO interested to explore R&D&C partnerships for technology evaluation and development in a range of crop platforms CSIRO undertaking techno-economic evaluation study to determine best deployment value propositions and alternative product strategies CSIRO interested to access expertise in supply chain logistics for biomass feedstock production, accumulation and processing (e.g. cellulosic ethanol experience).

37 Thank you ACKNOWLEDGEMENTS Surinder Singh James Petrie Thomas Vanhercke Anna El Tahchy Pushkar Shrestha Uday Divi Ben Leita Gonzalo Estavillo Qing Liu Craig Wood XueRong Zhou Phil Larkin Peter Nichols Peter Mansour Rob Defeyter Allan Green Research Director - Bioproducts T E allan.green@csiro.au CSIRO Food & Nutrition Flagship CSIRO Agriculture Flagship CSIRO Energy Flagship