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Production of a Biodegradable Plastic in the C4 Biomass Crop Sugarcane

Production of a Biodegradable Plastic in the C4 Biomass Crop Sugarcane. Richard McQualter; Research Fellow, AIBN, UQ. GREENHOUSE GASES. + CO 2. PHB. CO 2. Biomass crops. Linear polyesters produced in nature by bacteria Carbon storage compound Properties similar to polyester plastics

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Production of a Biodegradable Plastic in the C4 Biomass Crop Sugarcane

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  1. Production of a Biodegradable Plastic in the C4 Biomass Crop Sugarcane Richard McQualter; Research Fellow, AIBN, UQ.

  2. GREENHOUSE GASES

  3. + CO2 PHB CO2

  4. Biomass crops

  5. Linear polyesters produced in nature by bacteria Carbon storage compound Properties similar to polyester plastics Commercially produced by fermentation – small scale not cost competitive with petroleum derived plastics Polyhydroxyalkanoates

  6. Polyhydroxyalkanoates CH3  CH2 / CH2 CH3 / CH2 R CH3 Polyhydroxybutyrate Polyhydroxyalkanoate R = alkyl or functional groups Polyhydroxyvalerate Polyhydroxyhexanoate

  7. Properties of PHB • Water insoluble. • Good oxygen permeability. • Good ultra-violet resistance. • Soluble in chloroform and other chlorinated hydrocarbons. • Biocompatible. • Melting point 175°C., and glass transition temperature 2°C. • Tensile strength 40 MPa, close to that of polypropylene. • Sinks in water which facilitating its anaerobic biodegradation. • Nontoxic. • Less 'sticky' when melted, making it a potentially good material for clothing in the future

  8. Propylene PHB Butanol Maleic anhydride Crotonic acid Polypropylene Biofuel Polyester resins

  9. PHB synthesis in Cupriavidusnecator

  10. Subcellular sources of Acetyl-CoA CO2 Chloroplast Peroxisome fatty acid beta oxidation Isoprenoids Calvin cycle Aceto acetyl-CoA Acetyl-CoA Pyruvate Acetyl-CoA Acetyl-CoA Pyruvate Mitochondrion NADPH Pyruvate De novo fatty acid synthesis Acetyl-CoA Citrate cycle

  11. Peroxisomal PHB production 10 9 8 7 6 5 4 3 2 1 0 Tilbrook K, Gebbie L, Schenk PM, Poirier Y, Brumbley SM (2011) Peroxisomal polyhydroxyalkanoate biosynthesis is a promising strategy for bioplastic production in high biomass crops. Plant Biotechnology Journal 9:958-969

  12. Plastidic PHB production 10 9 8 7 6 5 4 3 2 1 0 Petrasovits LA, Purnell MP, Nielsen LK, Brumbley SM (2007) Production of polyhydroxybutyrate in sugarcane. Plant Biotechnol J 5:162-172 Purnell MP, Petrasovits LA, Nielsen LK, Brumbley SM (2007) Spatio-temporal characterization of polyhydroxybutyrate accumulation in sugarcane. Plant Biotechnol J 5:173-184

  13. Stronger promoters Promoters Cv - banana streak badnavirus Cab5 – Maize chlorophyl A/B binding protein R-Ubi – Rice polyubiquitin UbiABC – Maize polyubiquitin

  14. Cab-m5 promoter 10 9 8 7 6 5 4 3 2 1 0 Petrasovits LA, Zhao L, McQualter RB, Snell KD, Somleva MN, Patterson NA, Nielsen LK, Brumbley SM (2012) Enhanced polyhydroxybutyrate production in transgenic sugarcane. Plant Biotechnol J 10:569-578

  15. Reduce competition PhaA PhaB PhaC ACCase KASIII Fatty acid biosynthesis

  16. Chemical knockout of ACCase activity • Stops fatty acid biosynthesis and halts plant growth

  17. 0 µM 20 µM 100 µM Petrasovits LA, McQualter RB, Gebbie LK, Blackman DM, Nielsen LK, Brumbley SM (2013) Chemical inhibition of acetyl coenzyme A carboxylase as a strategy to increase polyhydroxybutyrate yields in transgenic sugarcane. Plant Biotechnology Journal n/a-n/a

  18. Chemical ripening of sugarcane

  19. Modified PHBPathway PhaA PhaB Km ~ 1.1 Km ~ 0.044 Xzyme Km ~ 0.07 PhaC ACCase Km ~ 0.09 KASIII Km ~ 0.02 Fatty acid biosynthesis

  20. PhaA Xzyme Wild type 10 9 8 7 6 5 4 3 2 1 0

  21. new glasshouse 3.9 1.6 0.1 3.5 0.3 7.0 5.2 0.5 12.0 0.6 5.6 10.1 old glasshouse 10.3 0.7 5.6 8.6 5.0 0.6 4.9 0.7 7.6 4.9 7.6 0.9 0.8 6.3 4.9 6.3 5.0 0.7 4.0 4.7 0.9 7B4 29 37

  22. Metabolix – increasing carbon assimilation in PHB producing switchgrass 5.24% DW PHB w/o = 2.55% Enhanced photosynthesis, starch content and plant growth compared to base PHB pathway 7.69% DW PHB w/o = 3.53%

  23. Factors facilitating increased PHB: Targeting to chloroplasts or peroxisomes Stronger promoter (Cab-m5) Chemical knockout of ACCase More efficient enzyme (Xzyme) Bifunctional enzyme – doubled PHB in switchgrass Round 1 Round 2 Round 3 0.6-0.8% 2.5% >6.5%

  24. TOPS • PHB • Electricity co-generation • 15% retained on field to prevent erosion • STEMS • Sugars • Industrial chemicals • Biofuel • Fibre

  25. Value add-on for sugar industry 115 % of trash and tops left on the fields for erosion prevention. 2Cost of collection of tops and trash, hauling to the processing plant, extraction and purification of the biopolymer. *Estimated at half of gross value of PHB at 10% dry weight.

  26. Future Directions Transfer technology to commercially important sugarcane varieties • Evaluate performance under field conditions • Find AgBiotech company to commercialise PHB producing sugarcane

  27. AIBN Lars Nielsen Lars Petrasovits Leigh Gebbie Deborah Blackman Acknowledgements Metabolix Kristi Snell Maria Somleva Funding BSES Ltd CRCSIIB ARC linkage Metabolix UNT Stevens Brumbley Metabolomics Australia Mark Hodson Manuel Plan

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