Zusammenfassung
Corynebacterium glutamicum is intensively used for the industrial
large-scale (fed-) batch production of amino acids, especially glutamate
and lysine. However, metabolic flux analyses based on C-13-labeling
experiments of this organism have hitherto been restricted to
small-scale batch conditions and 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 developed (E1 Massaoudi et al., Metab. Eng., submitted), in
which a 300-L production reactor and a 1-L Sensor Reactor are run in
parallel master/slave modus, thus enabling C-13-based metabolic flux
analysis to generate a series of flux maps that document large-scale
fermentation courses in detail. We describe the successful combination
of this technology with nuclear magnetic resonance (NMR) analysis,
metabolite balancing methods and a mathematical description of
C-13-isotope labelings resulting in a powerful tool for quantitative
pathway analysis during a batch fermentation. As a first application,
C-13-based metabolic flux analysis was performed on exponentially
growing, lysine-producing C. glutamicum MH20-22B during three phases of
a pilot-scale batch fermentation. By studying the growth, (co-)
substrate consumption and (by-) product formation, the similarity of the
fermentations in production and Sensor Reactor was verified. Applying a
generally applicable mathematical model, which included metabolite and
carbon labeling balances for the analysis of proteinogenic amino acid
C-13-isotopomer labeling data, the in vivo metabolic flux distribution
was investigated during subsequent phases of exponential growth. It was
shown for the first time that the in vivo reverse C-4-decarboxylation
flux at the anaplerotic node in C. glutamicum significantly decreased
(70\%) in parallel with threefold increased lysine formation during the
investigated subsequent phases of exponential growth. (C) 2003 Elsevier
Inc. All rights reserved.
Nutzer
