Effect of modifying metabolic network on poly-3-hydroxybutyrate biosynthesis in recombinant Escherichia coli

The regulatory mechanism for poly-3-hydroxybutyrate (PHB) biosynthesis in recombinant Escherichia coli is markedly different from that of Ralstonia eutropha (formerly, Alcaligenes eutrophus) since the former efficiently synthesizes PHB during growth without any nutrient limitation. To analyze how the central metabolic pathways should be balanced with pathways necessary for cell growth and PHB formation, a stoichiometric model was developed to predict the theoretical maximum PHB production capacity for different metabolic variants. Flux analysis results illustrated the importance of the availability of acetyl-CoA and NADPH for achieving the maximum yield of PHB. In order to examine whether the increased availability of the above substances can enhance PHB synthesis in recombinant E. coli, both genetic and environmental perturbations were attempted. Several E. coli K12 derivatives, namely, HMS174, TA3516 (pta^-/ack^-), and DF11 (pgi^-), were transformed with a plasmid which contains the native phb operon. The fermentation characteristics of these recombinant strains were studied and compared. In this study we examined the effects of intracellular acetyl-CoA accumulation, which may promote PHB synthesis in vivo, by perturbations induced from attenuation of acetate kinase and phosphotransacetylase (TA3516, blocked in the acetate pathway) and by cultivation of E. coli HMS174 on gluconate; it can convert gluconate to acetyl-CoA at a higher rate. The effects of intracellular accumulation of NADPH were investigated by introducing a perturbation induced from attenuation of phosphoglucose isomerase, which redirects the carbon flow to the pentose-phosphate (PP) pathway. Results from the analyses of these perturbations indicate that intracellular buildup of acetyl-CoA may not be able to promote PHB synthesis in vivo. On the other hand, since the biosynthesis of PHB in the pgi^- mutant strain can utilize the NADPH overproduced through the PP pathway, the growth of the pgi^- mutant on glucose was recovered, indicating that the overproduction of NADPH might be able to enhance PHB synthesis.