Metabolic engineering of central carbon metabolism in Escherichia coli: Improving the production of biomass and metabolites

The pathway for central carbon metabolism provides precursors for cell biosynthesis and metabolite synthesis. We investigated the metabolic engineering of the pathway of glycogen synthesis and degradation. In aerobic cultures in E. coli the uptake of nutrients occurs faster than the utilization of the precursors, in making biomass and energy. We decided to sequester the excess carbon in glycogen and devised vectors to degrade the sequestered glycogen.;When glycogen was sequestered, we observed changes in some of the secreted end-products. We observed that, after overproduction of glycogen, uptake of the previously secreted pyruvate was increased with respect to the control strain, and the CO;Comparison of cAMP levels with and without glycogen overproduction indicate a higher level in cAMP after glycogen is overproduced. There appears to be a tentative link, though not conclusive, between cAMP synthesis and glycogen synthesis pathway. cAMP is a global regulator of central carbon metabolism including many genes of the TCA cycle enzymes. By affecting the TCA flux, cAMP may be one of the causes behind the pleiotropic effects of glycogen overproduction and degradation.;This manipulation of the glycogen pathway yielded practically useful results. The metabolic engineering was done in an Escherichia coli mutant defective in acetate biosynthesis due to deletion of the acetate kinase and phosphotransacetylase genes. The sequestering of glycogen was achieved by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under the control of the tac promoter. If glycogen overproduction in the strain grown in complex medium was induced during late log-phase, biomass production increased by 15-20% relative to uninduced controls. When glycogen was sequestered and then degraded in E. coli cultures grown in minimal medium, by overamplifying the genes for glycogen synthesis and degradation, then glutamate production was increased almost 3-fold compared to the plasmid-free strain.