Metabolic engineering and NMR analysis of Bacillus subtilis and Escherichia coli

The goal of this research was to improve the properties of Bacillus subtilis and Escherichia coli for producing biochemical products. Specifically we aimed to reduce undesirable acid by-products when growth occurs in glucose-limiting medium. The approach relied on the rational design of biochemical reaction networks, known as Metabolic Engineering, as opposed using trail and error mutagenesis. We have focused our effort on (1) determining the optimal fluxes, which helps predict the maximal obtainable yields or theoretical yields of either biomass or desired metabolites, and (2) predicting the change in optimal flux pattern in response to a mutation strategy or to an environmental change. We have developed a metabolic engineering analytical infrastructure that integrates flux analysis and NMR experimental design. We have demonstrated the use of this analytical tool to design an NMR experiment that reveals flux distribution and regulatory control in B. subtilis and E. coli. Based on the theoretical analysis and NMR evidence, we pinpointed pyruvate kinase as the mutational target, and, through a collaboration with the University of Pittsburgh, constructed PYK-deficient B. subtilis and E. coli. We demonstrated that our metabolically engineered strains do not produce any organic acid as a by-product and consequently yield higher cell concentrations. In addition, our analysis indicated that the B. subtilis strain can be the potential host for the production of valuable and highly demanded products, e.g. folic acid. Finally, the overall utility of the analysis software has been addressed by developing user interfaces that allow for model alteration.