Verlag des Forschungszentrums Jülich
JUEL-4016
Anderlei, Britta
Untersuchungen zur rolle der Pyruvatcarboxylase als neuer anaplerotischer Reaktion in Escherichia coli
IV, 165 S., 2003
The expression of pyruvate carboxylase (Pyc) from Corynebacterium glutamicum
established a novel interconnection between glycolysis and tricarboxylic acid (TCA)
cycle in the central metabolism of 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 E. coli,
the pyc gene was introduced in an E. coli PEP carboxylase (ppc) deficient mutant,
which is devoid of any anaplerotic reaction during growth with glucose as sole carbon
source. The phenotype of the ppc mutant was suppressed by 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 E. coli cells. Pyc did not affect the exponential growth rate of the E. coli
wild type during aerobic cultivation with glucose, but accelerated growth with lactate
as sole carbon source.
To investigate the effects of Pyc on the availability of the intermediate
phosphoeno/pyruvate (PEP) in 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 ppc 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 E. coli aroB mutant by
30%, this indicates an increased PEP/pyruvate ratio. In a ppc 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 ppc 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 E. coli strains was significantly reduced. These results indicate
increased anaplerotic carbon fluxes to the TCA metabolism by Pyc.
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