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

The regulatory mechanism for poly-3-hydroxgbutyrate (PHB) biosynthesis in r ecombinant Escherichia coil is markedly different from that of Ralstonia eu tropha (formerly, Alcaligenes eutrophus) since the former efficiently synth esizes PHB during growth without any nutrient limitation. To analyze holy t he central metabolic pathways should be balanced with pathways necessary fo r cell growth and PHB formation, a stoichiometric model was developed to pr edict the theoretical maximum PHB production capacity for different metabol ic variants. Flux analysis results illustrated the importance of the availa bility of acetyl-CoA and NADPH for achieving the maximum yield of PHB. In o rder to examine whether the increased availability of the above substances can enhance PHB synthesis in recombinant E. coil, both genetic and environm ental perturbations were attempted. Several E. coil K12 derivatives, namely , HMS174, TA3516 (pta(-)/ack(-)), and DF11 (pgi(-)), were transformed with a plasmid which contains the native phl, operon. The fermentation character istics of these recombinant strains were studied and compared. In this stud y we examined the effects of intracellular acetyl-CoA accumulation, which m ay promote PHB synthesis in vivo, by perturbations induced from attenuation of acetate kinase and phosphotransacetylase (TA3516, blocked in the acetat e pathway) and by cultivation of E. coli BMS174 on gluconate; it can conver t gluconate to acetyl-CoA at a higher rate. The effects of intracellular ac cumulation of NADPH were investigated by introducing a perturbation induced from attenuation of phosphoglucose isomerase, which redirects the carbon h ow to the pentose-phosphate (PP) pathway. Results from the analyses of thes e perturbations indicate that intracellular buildup of acetyl-CoA may not b e able to promote PHB synthesis in vivo. On the other hand, since the biosy nthesis of PHB in the pgi(-) mutant strain can utilize the NADPH overproduc ed through the PP pathway, the growth of the pgi(-) mutant on glucose was r ecovered, indicating that the overproduction of NADPH might be able to enha nce PHB synthesis.