ENGINEERING CYANOBACTERIA FOR BIOFUEL PRODUCTION Ryan Hill, PhD candidate, Biochemistry.
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Transcript of ENGINEERING CYANOBACTERIA FOR BIOFUEL PRODUCTION Ryan Hill, PhD candidate, Biochemistry.
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
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)
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
The most important process of life
Transcription
Translation
DNA DNA+RNA polymerase
mRNA
mRNA+Ribosome
Protein X
Promoter
RBS
ORF
Terminator
Ribosome
Protein X assembly
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
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
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 J 2, 1525-1534
Putting it together – Why?
Synechocystis offers 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
Metabolic pathways
Clostridium beijerinckii
butanol pathway
Synechocystis poly-[hydroxybutyrate]
(PHB) pathway
PHB production is circadian controlled, i.e. turned on at night and shut down in the day
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
Synechocystis strains
pRH-ECT7 based: ∆phaEC::aph (kanamycin resistance) ∆phaEC::luxAB, aph
pRH-BT7b based: ∆phaAB::cat (chloramphenicol resistance) ∆phaAB::luxAB, cat
Testing the programs – ECT7::luxAB
0 2 4 6 8 10 12 14 16 18 20 22 240.0
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Normalised luciferase activity from ∆phaEC::luxAB, aph
Time, hours
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Light
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Testing the programs – BT7b::luxAB
0 2 4 6 8 10 120
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Normalised luciferase activity from ∆phaAB::luxAB, cat
Time, hours
Activ
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max
imum
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