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

Expression plasmids – ECT7

pRH-ECT7::luxAB, kan

Synechocystis strains

pRH-ECT7 based: ∆phaEC::aph (kanamycin resistance) ∆phaEC::luxAB, aph

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

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Normalised luciferase activity from ∆phaEC::luxAB, aph

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Testing the programs – BT7b::luxAB

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Normalised luciferase activity from ∆phaAB::luxAB, cat

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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

Acknowledgements

Department of BiochemistryAssoc. Prof. Julian Eaton-RyeAssoc. Prof. John Cutfield

Funding-Department of Biochemistry-OERC-University of Otago