Verlag des Forschungszentrums Jülich
JUEL-3824
Krämer, Marco
Untersuchungen zum Einfluß erhöhter Bereitstellung von Erythrose-4-Phosphat und Phosphoenolpyruvat auf den Kohlenstofffluss in den Aromatenbiosyntheseweg von Escherichia coli
131 S., 2000
In Escherichia coli wild-type strains carbon flux into the aromatic amino acid pathway is limited by the intermediates of central metabolism, erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP). The effects of increased supply of E4P and PEP on the carbon flux into the aromatic amino acid pathway were investigated in aroB strains which were deregulated in the aromatic amino acid pathway by introduction and expression of a gene of a feedback-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP)-synthase (aroFfbr). Carbon flux was quantified by measuring excreted DAHP.
For increasing availability of E4P expression of the plasmiden coded genes transaldolase (talB), transketolase (tktA), and the combination of transaldolase and transketolase were increased in combination with increased expression of aroFfbr. Increased expression of talB increased carbon flux into the aromatic amino acid pathway in correlation with higher specific activities of DAHP-synthases.
For increasing availability of PEP the phosphoenolpyruvate-phosphotransferase-system (PTS) was disrupted. Increased expression of the plasmiden coded genes of aroFfbr, glucose facilitator (glf) and glucokinase (glk), both of Zymomonas mobilis increased carbon flux into the aromatic amino acid pathway. For increasing availability of both PEP and E4P, the genes tktA, talB and the operon of tktA and talB were combined with the previous genetic system. Overexpression of talB led to a further increase of carbon flux into the aromatic amino acid pathway. Increased expression of talB correlated with even more higher increased specific activities of DAHP-synthases.
A new glucose uptake and phosphorylation system was established in E. coli cells in order to increase availability of E4P and PEP by circumventing carbon flux from glycolysis into the pentose phosphate pathway After introducing the genes of the glucose facilitator (glf), the glucose dehydrogenase of Bacillus megaterium (gdhIV) and a homologous gluconate kinase (gntK) growth of an Escherichia coli mutant without glucose uptake and phosphorylation system occurred. However expression of glf, gdhIV, gntK and aroFfbr in a PTS - - strain led to a low carbon flux into the aromatic amino acid pathway.
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