Renewable green alternatives to fossil fuels need to be sought in order to address the challenges of environmental and energy crises. Up until now, ethanol has been the major biofuel. Geobacillus thermoglucosidans is a thermophilic bacterium that is capable of producing bioethanol in an industrial setting at high temperatures and is capable of metabolizing pentoses and hexoses commonly found in lignocellulosic biomass. Due to these attractive properties, the aim of this work has been to construct a toolbox of genetic components to develop G. thermoglucosidans as as the leading thermophile chassis for synthetic biology and metabolic engineering. The toolbox is composed of shuttle vectors that have higher transformation efficiencies than previous existing vectors and are modular, where the presence of restriction sites separating each of the components allows users to exchange parts easily and efficiently. Also included in the toolbox are the fluorescent reporters sfGFP, mCherry and BsFbFP that will permit the characterization of promoters. As a proof-of-principle application to demontrate the effectivity of the toolbox for the production of valuable compounds, this work explores the production of isobutanol by the thermophile bacteria Geobacillus thermoglucosidans. Isobutanol is a higher chain alcohol that is a significantly better fuel molecule than ethanol, both for energy content and infrastructure compatibility. The Geobacillus host was able to produce isobutanol in amounts of around 50 mg/L via the conversion of isobutyryl-CoA to isobutyraldehyde by an (ALDH) and from isobutyraldehyde to isobutanol by an alcohol dehydrogenase (ADH). It was observed that supplementing the growth medium with an intermediate of the valine biosynthesis pathway, 2-ketoisovalerate, resulted in the production of isobutanol and overexpressing ALDH increased the isobutanol titres.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:712835 |
| Date | January 2014 |
| Creators | MartÃnez-Klimova, Elena |
| Contributors | Ellis, Tom ; Leak, David |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/45409 |
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