Hermann Sahm, Lothar Eggeling, Albert A. de Graaf
Pathway Analysis and Metabolic Engineering in Corynebacterium glutamicum
The Gram-positive bacterium Corynebacterium glutamicum
is used for the industrial production of amino
acids, e.g. of L-glutamate and L-lysine. During the last
15 years, genetic engineering and amplification of
genes have become fascinating methods for studying
metabolic pathways in greater detail and for the construction
of strains with the desired genotypes. In
order to obtain a better understanding of the central
metabolism and to quantify the in vivo fluxes in C.
glutamicum, the [13C]-labelling technique was combined
with metabolite balancing to achieve a unifying
comprehensive pathway analysis. These methods can
determine the flux distribution at the branch point between
glycolysis and the pentose phosphate pathway.
The in vivo fluxes in the oxidative part of the pentose
phosphate pathway calculated on the basis of intracellular
metabolite concentrations and the kinetic
constants of the purified glucose-6-phosphate and
6-phosphogluconate dehydrogenases determined in
vitro were in full accordance with the fluxes measured
by the [13C]-labelling technique. These data indicate
that the oxidative pentose phosphate pathway in
C. glutamicum is mainly regulated by the ratio of
NADPH/NADP concentrations and the specific activity
of glucose-6-phosphate dehydrogenase. The carbon
flux via the oxidative pentose phosphate pathway
correlated with the NADPH demand for L-lysine synthesis.
Although it has generally been accepted that phosphoenolpyruvate
carboxylase fulfills a main anaplerotic
function in C. glutamicum, we recently detected
that a biotin-dependent pyruvate carboxylase exists
as a further anaplerotic enzyme in this bacterium. In
addition to the activities of these two carboxylases
three enzymes catalysing the decarboxylation of the
C4 metabolites oxaloacetate or malate are also present
in this bacterium. The individual flux rates at this
complex anaplerotic node were investigated by using
[13C]-labelled substrates. The results indicate that
both carboxylation and decarboxylation occur simultaneously
in C. glutamicum so that a high cyclic flux of
oxaloacetate via phosphoenolpyruvate to pyruvate
was found.
Furthermore, we detected that in C. glutamicum
two biosynthetic pathways exist for the synthesis of
DL-diaminopimelate and L-lysine. As shown by NMR
spectroscopy the relative use of both pathways in vivo
is dependent on the ammonium concentration in the
culture medium. Mutants defective in one pathway are
still able to synthesise enough L-lysine for growth, but
the L-lysine yields with overproducers were reduced.
The luxury of having these two pathways gives C. glutamicum
an increased flexibility in response to changing
environmental conditions and is also related to the
essential need for DL-diaminopimelate as a building
block for the synthesis of the murein sacculus.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 381, 09/2000
Pages: 899 - 910
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