ENGINEERING
CYANOBACTERIA FOR
BIOFUEL PRODUCTION
Ryan Hill, PhD candidate, Biochemistry
Life is complex
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Metabolic engineers write “software” for living
systems
Living things are complex machines
Bacteria are essentially self-replicating micromachines
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)
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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
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Plasmid: Small circular hoop of DNA, “Mini
chromosome” encoding1-10 genes, 3-50kbp
The most important process of life
Transcription
mRNA
Promoter
DNA
DNA+RNA polymerase
ORF
RBS
Terminator
Protein X assembly
Protein X
mRNA+Ribosome
Translation
Ribosome
Synechocystis sp. PCC 6803
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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
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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?
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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
Synechocystis poly[hydroxybutyrate]
(PHB) pathway
PHB production is circadian
controlled, i.e. turned on at
night and shut down in the
day
Metabolic pathways
Clostridium beijerinckii
butanol pathway
Plasmids
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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
Expression plasmids – ECT7
pRH-ECT7::luxAB, kan
Synechocystis strains
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pRH-ECT7 based:
 ∆phaEC::aph
(kanamycin resistance)
 ∆phaEC::luxAB, aph
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pRH-BT7b based:
 ∆phaAB::cat
(chloramphenicol resistance)
 ∆phaAB::luxAB, cat
PHB Detection
Wildtype
∆phaAB::cat
∆phaEC::aph
∆phaAB::luxAB, cat
∆phaEC::luxAB, aph
Testing the programs – ECT7::luxAB
Normalised luciferase activity from ∆phaEC::luxAB, aph
110.0
60
100.0
50
90.0
80.0
60.0
30
50.0
40.0
20
30.0
20.0
10
10.0
0.0
0
0
2
4
6
8
10
12
Time, hours
14
16
18
20
22
24
Light, umol.m-2.s-1
Activity, % maximum
40
70.0
Testing the programs – BT7b::luxAB
Normalised luciferase activity from ∆phaAB::luxAB, cat
110
60
100
50
90
80
Activity, % maximum
40
70
60
30
50
40
20
30
20
10
10
0
0
0
2
4
6
Time, hours
8
10
12
Summary
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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
Acknowledgements
Department of Biochemistry
Assoc. Prof. Julian Eaton-Rye
Assoc. Prof. John Cutfield
Funding
-Department of Biochemistry
-OERC
-University of Otago