Development of metabolic engineering strategies for the enhanced production of metabolites

Abstract

Metabolic engineering studies for the production of various metabolites in Escherichia coli have been carried out. For these tasks, metabolic flux analysis (MFA) technique was applied to identify metabolic condition of recombinant E. coli strains. At first, a pfl ldhA double mutant E. coli strain NZN111 was used to produce succinic acid by overexpressing the E. coli malic enzyme. To have an insight into the intracellular metabolism, MFA was carried out. From the result, it was predicted that supplying additional reducing power could enhance succinic acid production, and more reduced carbon substrate sorbitol was thus examined for the possibility of matching the potential during succinic acid production. When NZN111 (pTrcML) was cultured in LB medium containing 20 g/L sorbitol under CO2 atmosphere, 10 g/L of succinic acid was produced, and the apparent yield of succinic acid was 1.1 g succinic acid/g sorbitol. Secondly, the metabolic network of E. coli was constructed, and was used to simulate the distribution of metabolic fluxes in recombinant E. coli producing poly(3-hydroxybutyrate) [P(3HB)]. It was notable to find from MFA result that the Entner-Doudoroff (ED) pathway flux increased significantly under P(3HB) accumulating condition. The P(3HB) content obtained with KEDA (pJC4), which is defective in the activity of Eda, was lower than that obtained with its parent strain. The reduced P(3HB) biosynthetic capacity of KEDA (pJC4) could be restored by the coexpression of the E. coli eda gene, which proves the important role of ED pathway on P(3HB) synthesis in recombinant E. coli as predicted by metabolic flux analysis. Thirdly, a new strategy, metabolic flux profiling and clustering, was proposed and applied to systematically study the variation of metabolic fluxes in E. coli under metabolite production conditions. Through those analyses, I was able to predict the real achievable maximum yields of metabolites rather than theoretical maximum yields, which ca...