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Title: Metabolic engineering of Escherichia coli for the biosynthesis of astaxanthin
Author: Scaife, Mark Aden
ISNI:       0000 0001 3554 6722
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2008
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Carotenoids hold great promise due to their numerous health promoting properties. Of the plethora of carotenoids isolated, astaxanthin is one of the most intensively investigated. Astaxanthin can be synthesised by a number of organisms, however these are often not amenable to current industrial production methods, and those that are produce low or impure yields. In this thesis, data are presented' regarding the development of an Escherichia coli based astaxanthin production system that is able to rapidly accumulate industrially relevant quantities of highly pure astaxanthin. In pursuit of this, the genomes of five cyanobacterial strains are screened in order to identify putative genes involved in the final stages of astaxanthin biosynthesis, specifically in the conversion of f3-carotene to astaxanthin. Subsequently the sixteen putative genes identified are functionally characterised, via co-expression in E. coli, and investigated with respect to their capacity to function in astaxanthin biosynthesis. This reveals that all twelve f3-carotene ketolase genes identified are functional, as are two of the four f3-carotene hydroxylase genes. However, only the CrtW type f3. carotene ketolase genes are able to participate in astaxanthin biosynthesis. The best performing elements, with respect to astaxanthin biosynthesis, are combined and employed in the creation of a number of E. coli based astaxanthin production strains. Subsequent studies focus on metabolic restrictions within this system, employing heterologous gene expression and directed evolution methods, as well as a novel PCR based cloning method, in the creation of an E. coli production strain capable of the synthesis of 4.7 mg L-1 astaxanthin in a 24 hour period, at> 90% purity. Further, this study identifies a range of restrictions within the system, and presents methods by which future work could negotiate these, allowing the development of an industrially competitive production strain.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available