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Engineering cyanobacteria for biofuel production

Engineering cyanobacteria for biofuel production

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Engineering cyanobacteria for biofuel production

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  1. Engineering cyanobacteria for biofuel production Ryan Hill, PhD candidate, Biochemistry

  2. Life is complex • Metabolic engineers write “software” for living systems • Living things are complex machines • Bacteria are essentially self-replicating micro-machines • Components are at the nano-/molecular scale • Life is “programmable” – Software is written in DNA and executed by the cell machinery

  3. Key Molecules and enzymes Key Molecules • DNA : Deoxyribonucleic Acid. One very large molecule (3Mbp-10+Gbp). Master copy • mRNA: messenger Ribonucleic Acid. Lots of small molecules (1-10kbp). Working copies • Enzyme: Amino acids. Proteins that catalyze chemical reaction. Molecular machines. Key Enzymes: • RNA Polymerase: Molecular machine that creates mRNA instructions from DNA templates (photocopier) • Ribosome: Molecular machine that reads mRNA and builds an enzyme from the instructions encoded (robot assembler)

  4. Gene: A region of DNA that encodes all the information necessary for producing an enzyme • Promoter: Region of gene that promotes transcription • Terminator: Stops transcription • RBS: Ribosomal binding site • ORF: Open reading frame. Region of a gene that encodes the enzyme information read by the ribosome • Plasmid: Small circular hoop of DNA, “Mini chromosome” encoding1-10 genes, 3-50kbp

  5. The most important process of life Transcription mRNA DNA DNA+RNA polymerase Promoter ORF RBS Terminator Protein X Protein X assembly mRNA+Ribosome Ribosome Translation

  6. Synechocystis sp. PCC 6803 • Single cell bacterium (cyanobacterium) • Photosynthetic – fixes carbon dioxide • Genome sequenced (3.5Mbp), well understood • Genome can be easily and precisely modified

  7. Butanol – Ethanols’ big brother • Comparable to petroleum 91-96 fuels • Compatible with current infrastructure • Compatible with current vehicles/engines • BUT current bio-production is inefficient • Clostridium beijerinckii or C. acetobutylicum • Acetone-Butanol-Ethanol (ABE) fermentation Ethanol Butanol

  8. The many uses of butanol “Until around 2005, butanol was only considered to be a bulk chemical precursor for production of acrylate and methacrylate esters, glycol ethers, butyl acetate, butylamines, and amino resins. Their use is manifold: production of adhesives/scalants, alkaloids, antibiotics, camphor, deicing fluid, dental products, detergents, elastomers, electronics, emulsifiers, eye makeup, fibers, flocculants, flotation aids (e.g., butyl xanthate), hard-surface cleaners, hormones and vitamins, hydraulic and brake fluids, industrial coatings, lipsticks, nail care products, paints, paint thinners, perfumes, pesticides, plastics, printing ink, resins, safety glass, shaving and personal hygiene products, surface coatings, super absorbents, synthetic fruit flavoring, textiles, as mobile phases in paper and thin-layer chromatography, as oil additive, as well as for leather and paper finishing” Durrie (2007) Biotechnol J2, 1525-1534

  9. Putting it together – Why? • Synechocystisoffers several advantages: • Do not require a feedstock (arable land), it makes it own • Grows in water • There is no processing of biomass • Majority of fixed carbon converted to bio-fuel • The growth procedure is also a butanol extraction procedure (gas stripping) • A near pure stream of butanol should be achievable directly from the bioreactor

  10. Metabolic pathways Synechocystis poly-[hydroxybutyrate] (PHB) pathway Clostridium beijerinckii butanol pathway PHB production is circadian controlled, i.e. turned on at night and shut down in the day

  11. Plasmids • Two base plasmids: • pRH-ECT7 – Knock-out of phaEC, inserts ORFs under control the phaEC promoter/RBS, uses T7 terminator • pRH-BT7b – Knock-in extra ORF(s) onto the end of the phaAB mRNA (after phaB), has RBS from psbA2 gene, uses T7 terminator

  12. Expression plasmids – ECT7

  13. pRH-ECT7::luxAB, kan

  14. Synechocystis strains • pRH-ECT7 based: • ∆phaEC::aph(kanamycin resistance) • ∆phaEC::luxAB, aph • pRH-BT7b based: • ∆phaAB::cat (chloramphenicol resistance) • ∆phaAB::luxAB, cat

  15. PHB Detection Wildtype ∆phaAB::cat ∆phaEC::aph ∆phaAB::luxAB, cat ∆phaEC::luxAB, aph

  16. Testing the programs – ECT7::luxAB

  17. Testing the programs – BT7b::luxAB

  18. Summary • Plasmids pRH-ETC7 and –BT7b constructed • Modification of PHB metabolism doesn’t damage Synechocystsis • pRH-ECT7 successfully knocks out PHB production • pRH-BT7b successfully does not damage PHB production • Both pRH-ECT7 and –BT7b work as intended when using luciferase as a reporter

  19. Acknowledgements Department of Biochemistry Assoc. Prof. Julian Eaton-Rye Assoc. Prof. John Cutfield Funding -Department of Biochemistry -OERC -University of Otago