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Title: The acetone-butanol-ethanol pathway of Clostridium acetobutylicum
Author: Wang, Hengzheng
ISNI:       0000 0004 7428 7956
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2014
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Biobutanol is superior biofuel to ethanol. However, the yield of butanol from Clostridium acetobutylicum is not ideal. Yield improvements will require a better understanding of the Acetone-Butanol-Ethanol (ABE) fermentation pathway. One approach to identify bottlenecks would be to differentially express the genes encoding key enzymes, and assess the consequences. This strategy requires either the use of promoters of varying strengths, or ideally, the use of a tightly regulated inducible promoter system in which the level of expression is inducer, dose dependent. To implement this strategy there is a need to identify the key genes to be targeted. Accordingly, the phenotypic consequences of ClosTron insertions in key genes of the ABE pathway (adhE, ctfAB and ptb) were confinned by complementation. This was unsuccessful in the case of the ptb mutant due to a secondary mutation in tlll. A library of promoters was isolated with a range of ’strengths’ and the suitability of various repOlier genes examined, including the gusA and cpg genes, as well as various genes encoding fluorescent proteins. They all, proved inappropriate, and the catP gene was eventually used routinely. It was used to test two inducible systems, one based on IPTG and the addition of a lacO operator to the promoter, and the other based on a lactose inducible system (BgaRIBgaL) from Clostridium per./i’ingens. Whilst both functioned in a dose dependent manner when on a plasmid, only the latter functioned when integrated into the genome. To overcome the effects of lower gene dosage when in the genome, an orthogonal expression system based on either BotR (Clostridium botulinum) or TcdR (Clostridium difficile) was successfully implemented. Attempts to make in-frame deletions in various genes (adhE, adc and ptbibuk) were unsuccessful, despite testing a number of different strategies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available