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A metabolic engineering approach to examine polyketide production by Saccharopolyspora erythraea

Ushio, Misti; (2003) A metabolic engineering approach to examine polyketide production by Saccharopolyspora erythraea. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The objective of this thesis was to use a metabolic engineering and modelling approach to evaluate the effect of genetic modifications in primary metabolism on secondary metabolite production in Saccharopolyspora erythraea. The thesis investigated 1) the physiology of wild-type and genetically modified S. erythraea strains studied in batch culture under various nutrient conditions, 2) the physical and antimicrobial properties of red pigments produced by S. erythraea, 3) the capabilities of metabolic flux analysis to provide useful predictions of metabolic engineering targets, and 4) the genetic engineering tools to manipulate the primary metabolism of S. erythraea. The physiology of S. erythraea, in particular the relationship between organic acid overflow metabolism and polyketide synthesis, was studied. Using S. erythraea physiology data and metabolic modelling predictions, overexpression of α-ketoglutarate dehydrogenase was selected as a metabolic engineering target. The aim was to determine if α-ketoglutarate excreted outside the cell could be re-channeled into primary metabolism to increase production of erythromycin and decrease red pigment synthesis. To this end, a new S. erythraea strain with an overexpressed heterologous α-ketoglutarate dehydrogenase was constructed and tested. The new strain was able to decrease α-ketoglutarate excretion, but did not produce greater amounts of erythromycin. Even though erythromycin levels were not increased this research could be considered as a first step to rational strain development for erythromycin production. It could also be regarded, in a greater sense, as proof of concept to use of the S. erythraea sequence data to identify genes of interest, and to genetically manipulate the central metabolism of S. erythraea. The results from this thesis justify research efforts to be continued in three different areas. The first is to continue strain improvement for erythromycin over-production. The second is to understand how to control organic acid excretion. The third is red pigment characterization.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: A metabolic engineering approach to examine polyketide production by Saccharopolyspora erythraea
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Polyketides
URI: https://discovery.ucl.ac.uk/id/eprint/10100518
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