Metabolic engineering and flux analysis of Escherichia coli
Doctor of Philosophy
The advances in molecular biology, genome sequencing and mathematical tools have led to growing interest in metabolic engineering, the goal of which is to redirect metabolic fluxes for industrial and medical purposes. Escherichia coli was chosen as the model system for several reasons: the ease of genetic manipulation, the wealth of genetic information, its capacity for rapid growth, and the availability of inexpensive and standardized cultivation protocols. The effects of precise perturbation in the central metabolic pathway of E. coli with an emphasis on the competition at the pyruvate node was investigated using recombinant DNA technology to create the changes and metabolic flux analysis to quantify the results. First, the effects of competition at the pyruvate node was studied by two modifications of central metabolism, the heterologous expression of the Bacillus subtilis acetolactate synthase (ALS) and the removal of the major acetate production pathway. This study was primarily a continuation of the application of metabolic engineering strategies to increase recombinant protein production through acetate reduction. The next study involved the effect of an additional mutation at the nuo gene encoding an NADH dehydrogenase recently mapped near the genes (ack4-pta ) controlling the acetate production pathway. In the earlier studies involving strains deficient in ackA-pta gene, lactate instead of acetate or ethanol was the major fermentation product. A natural extension of this project was to examine the effects of deficiency and overexpression of the lactate dehydrogenase (LDH) enzyme responsible for lactate production pathway. Next, the role of intermediate pool levels, specifically pyruvate, on redistribution of carbon flux and partitioning at the pyruvate node was examined. Sensitivity or flux control coefficients were calculated to gain further insight into dynamics of the overall reaction network. Finally, the effects of different enzyme properties on metabolic patterns were explored through the integration and expression of ALS from Klebsiella pneumoniae and a comparison with the B. subtilis ALS.
Microbiology; Biochemistry; Chemical engineering