Dissertation / PhD Thesis/Book

PreJuSER-24439

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Untersuchungen zur Rolle der Pyruvatcarboxylase als neuer anaplerotischer Reaktion in $\textit{Escherichia coli}$

Anderlei, B. (Corresponding author)FZJ*

2002
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich

Please use a persistent id in citations: http://hdl.handle.net/2128/90

Report No.: Juel-4016

Abstract: The expression of pyruvate carboxylase (Pyc) from $\textit{Corynebacterium glutamicum}$ established a novel interconnection between glycolysis and tricarboxylic acid (TCA) cycle in the central metabolism of $\textit{E. coli}$. The effects of this measure were investigated in the present work. In order to investigate whether the heterologous Pyc was functionally active in $\textit{E. coli}$, the $\textit{pyc}$ gene was introduced in an $\textit{E. coli}$ PEP carboxylase ($\textit{ppc}$) deficient mutant, which is devoid of any anaplerotic reaction during growth with glucose as sole carbon source. The phenotype of the $\textit{ppc}$ mutant was suppressed by $\textit{pyc}$ in a IPTG- and biotin- dependent manner without impairment of cell growth. With a discontinuous, coupled assay system Pyc activity of 230 mU/mg protein was detected in recombinant $\textit{E. coli}$ cells. Pyc did not affect the exponential growth rate of the $\textit{E. coli}$ wild type during aerobic cultivation with glucose, but accelerated growth with lactate as sole carbon source. To investigate the effects of Pyc an the availability of the intermediate phosphoenolpyruvate (PEP) in $\textit{E. coli}$, the novel reaction was introduced into a mutant with an inactive 3-dehydroquinate synthase gene (aroB). This indicator strain accumulates the first intermediate of the aromatic amino acid biosynthesis pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), the condensation product of PEP and erythrose-4-phosphate. DAHP is excreted into the culture supernatant and was detected as dephosphorylated DAH. Hence, the DAH production of aroB mutants gives information about the carbon flux into the shikimate pathway and the availability of PEP. Pyc was expressed as second anaplerotic reaction besides Ppc and also in a $\textit{ppc}$ $\textit{aroB}$ double mutant. To generate comparable process conditions for every tested strain, all fermentations were performed aerobically in stirred bioreactors with pH control in a fed batch mode. The expression of Pyc enhanced the substrate turnover of an $\textit{E. coli}$ $\textit{aroB}$ mutant by 30%, this indicates an increased PEP/pyruvate ratio. In a$\textit{ppc}$ $\textit{aroB}$ double mutant, Pyc improved the availability of PEP for the shikimate pathway by raising the DAH yield from 5 to 12% of the carbon consumed. A $\textit{ppc}$ $\textit{aroB}$ double mutant with Pyc produced more than 80 mM glutamate, which corresponds to one third of the carbon consumed. Furthermore, the acetate formation of Pyc expressing $\textit{E. coli}$ strains was significantly reduced. These results indicate increased anaplerotic carbon fluxes to the TCA metabolism by Pyc.

Note: Record converted from VDB: 12.11.2012
Note: Düsseldorf, Univ., Diss., 2002

---

Contributing Institute(s):

1. Biotechnologie 1 (IBT-1)

Research Program(s):

1. Biotechnologie (L02)

Appears in the scientific report 2002

---

Database coverage:

---