Verlag des Forschungszentrums Jülich
JUEL-4103
The flux analysis showed that the in vivo reverse C4-decarboxylation flux at the anaplerotic
node significantly decreased (70%) in parallel with threefold deceased lysine formation
during three investigated phases of exponential growth in batch fermentation. In a lysine
production phase during a fed-batch fermentation, in which no biomass synthesis was
observed, the reverse C4-decarboxylation flux was almost completely suppressed. These
results indicate that the reverse flux is counteractive to the production of lysine, because the
activity of this flux has an influance on the intracellular concentration of the lysine precursor
oxaloacetate.
To date, little information is available on the significance of the phosphoenolpyruvate (PEP)
carboxykinase, the most important enzyme of the reverse C4-decarboxylation flux. Therefore,
a PEP carboxykinase (pck)-deficient strain of C. glutamicum was constructed and tested on
different carbon sources and substrate mixtures in comparison to the wildtype strain . The
growth behaviour and the substrate consumption showed that the elimination of the PEP
carboxykinase activity has no effect on the utilization of different sugars as sole carbon
source. In contrast, additional supplementation of acetate or lactate to a culture medium with
glucose, sucrose or fructose lead to a decreased growth of the pck mutant. These results
show that acetate and lactate inhibit the PEP-dependent phosphotransferase system (PTS),
which is responsible for the uptake of several sugars in C, glutamicum .
Drysch, André
Intrazelluläre Flußquantifizierung unter instationären Wachstumsbedingungen und Mischsubstratverwertung in Corynebacterium glutamicum
110 S., 2003
Corynebacterium glutamicum is intensively used for industrial large-scale (fed-) batch
production of lysine. However, metabolic flux analyses based on 13C-labeling experiments of
C. glutamicum have hitherto been restricted to small-scale batch conditions and stationary
carbon-limited chemostat cultures and are therefore of questionable relevance for industrial
fermentations. To lever flux analysis to the industrial level, a novel sensor reactor approach
was combined with nuclear magnetic resonance (NIVIR) analysis, metabolite balancing
methods and the mathematical description of 13C-isotope labeling to obtain a series of
intracellular carbon flux maps documenting the changes of intracellular flux distributions
during (fed-) batch fermentation processes. Experimental 13C labeling patterns of proteinogenic
amino acids and cytoplasmic metabolites were used to yield the intracellular metabolic
fluxes.
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