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dc.contributor.advisorEaton-Rye, Julian
dc.contributor.authorHill, Ryan Edward
dc.date.available2013-10-14T20:29:58Z
dc.date.copyright2013
dc.identifier.citationHill, R. E. (2013). The metabolic engineering of Synechocystis sp. PCC 6803 for production of n-butanol (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/4330en
dc.identifier.urihttp://hdl.handle.net/10523/4330
dc.description.abstractThe cyanobacterium Synechocystis sp. PCC 6803 is an attractive target for engineering novel metabolic pathways for the synthesis of useful compounds directly from CO2 as it is a naturally transformable, oxygenic photoautotroph and the genome has been sequenced. The compound n-butanol is a potential bio-fuel for direct replacement of petroleum, with little to no adjustment of the current infrastructure, as n-butanol has similar fuel characteristics to petroleum. To accomplish the aim, an integrative expression system was developed consisting of two plasmids, which integrated at the phaAB and phaEC loci. Integration at the phaEC site eliminated a competing pathway, polyhydroxybutyrate (PHB) biosynthesis. The two plasmids were used to introduce foreign genes into the genome under the control of promoters PphaA and PphaE. The expression system was validated and analysed using a luciferase reporter enzyme. The promoters were found to express under circadian rhythm, expression was increased in the dark and repressed in the light. In addition, under phosphate limitation the luciferase expression was increased three-fold and became constitutively expressed. Expression was strongly up-regulated in the stationary compared to the logarithmic growth phase. The genes for n-butanol biosynthesis pathway from Clostridium beijerinckii were introduced into Synechocystis sp. PCC 6803 under control of the PphaA and PphaE promoters; however, no butanol could be detected above background levels. Enzyme assays of the cell lysate showed that two of the enzymes activities could not be detected, indicating either the enzymes were inactive or being expressed below limits of detection. Replacement of the C. beijerinckii Bcd-complex with crotonyl-CoA reductase (Ccr) from Streptomyces collonius, along with codon optimisation of the remaining C. beijerinckii genes, generated strain SynRH-10, which synthesised 36 μg/L of culture in eight days and had a peak output of 14 μg/day/L of culture. The aldehyde dehydrogenase enzyme was identified as a potential bottleneck and will require replacement to improve n-butanol output. The program Precog was developed to identify additional novel pathways from MetaCyc reaction and compound data. Several novel pathways were identified, in doing so it also identified Ccr as an additional putative bottleneck in the pathway. An engineering strategy and new pathway was proposed, based on the results of this research and from other studies, which could generate a strain capable of synthesising milligram quantities per litre of culture.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectcyanobacteria
dc.subjectSynechocystis
dc.subjectbutanol
dc.subjectmetabolic
dc.subjectengineering
dc.subjectbiofuel
dc.titleThe metabolic engineering of Synechocystis sp. PCC 6803 for production of n-butanol
dc.typeThesis
dc.date.updated2013-10-14T07:49:14Z
dc.language.rfc3066en
thesis.degree.disciplineBiochemistry
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.openaccessOpen
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