A metabolic engineering approach to study overflow metabolism in E. coli
Aerobic fermentations of Escherichia coli grown with glucose accumulate organic acids in the culture medium. These by-products of glucose metabolism are synthesised and excreted when the glucose uptake rate is greater than its conversion to biomass and carbon dioxide. Acetate is the most abundant overflow metabolite produced in aerobic cultures of E. coli. Its production is strain and media specific, being greatest in dense nutrient-rich cultures. In industrial fermentations, E. coli is widely employed for production of recombinant proteins. However, acetate excretion reduces process efficiency by lowering cell growth rate, and decreasing the amount of substrate carbon converted to recombinant protein product. This thesis describes the construction of three plasmid vectors designed to express antisense RNA targeted against phosphotransacetylase and acetate kinase, which convert acetyl CoA to acetate. Antisense RNA was used as a metabolic engineering tool in this study, to enable examination of the central carbon flux distribution before, during and after enzyme downregulation. The aim was to decrease expression of these enzymes in E. coli, and thus decrease acetate production. E. coli MG1655 was transformed with a) a plasmid construct encoding an antisense RNA fragment, b) two compatible plasmids encoding different antisense RNA fragments. The resulting strains were cultivated in a 2L bioreactor, and the effect of antisense RNA expression evaluated. Assays to monitor the enzyme activities of phosphotransacetylase and acetate kinase were conducted, along with metabolite analysis to determine organic acid excretion profiles. Flux balance analysis, which is a method of modelling metabolism, was applied to the central carbon pathways in E. coli to provide insight into the partitioning of internal carbon fluxes. Information about the mechanisms used by E. coli to cope with partial shutdown of the acetate synthesis pathway was gained by comparing the flux distribution of a control strain with an antisense RNA-expressing strain.