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Title: Proteomic investigation of an Escherichia coli terpene production factory : prospects for metabolic engineering
Author: Robert, Faith Owabhel
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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Natural products, produced as secondary metabolites by plants and microbes, have been used for thousands of years to treat human ailments and still form the basis of drug discovery efforts. Terpenes form the largest category of natural products. Terpenes such as the antimalarial compound artemisinin and the anticancer compound taxol are potent medicines. Furthermore, terpenes are active ingredients in perfumes, nutraceuticals and biofuels. The fact that these products are produced in very small quantities in their natural host, from sources that are potentially exhaustible and prone to environmental fluctuations, have fuelled interest in sustainable, affordable and environmentally friendly means of production. Genetic engineering has proved an invaluable tool in producing considerable amounts of terpenes in heterologous hosts such as Escherichia coli and yeast. In this thesis, genetic/metabolic engineering and –omics strategies have been applied for the more efficient production of the anti-tumour compound β-elemene. Heterologous expression of sesquiterpene synthase from Nostoc sp. PCC 7120 in E. coli resulted in the successful production of β-elemene. Final β-elemene titres of up to 178 mg/L were quantified using targeted metabolomics strategies. Simultaneous monitoring of regulation of metabolic flux in recombinant E. coli producing β-elemene was carried out by applying isobaric tags for relative and absolute quantification (iTRAQ), a proteomics tool for relative quantification. A systematic evaluation of recombinant E. coli strains identified pathways that can be further optimised using metabolic engineering for commercially viable synthesis of β-elemene. This is the first report of an iTRAQ aided metabolic engineering study of terpene production in E. coli. This study demonstrates the importance of systems level –omic analyses in shedding light on bottlenecks that prevent commercially viable synthesis of highly beneficial natural products like terpene with E. coli as a biological chassis.
Supervisor: Wright, Phillip Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available