Serial flux mapping of Corynebacterium glutamicum during fed-batch L-lysine production using the sensor reactor approach.

Using our recently developed sensor reactor approach, lysine-producing, nongrowing Corynebacterium glutamicum MH20-22B cells were subjected to serial (13)C-labeling experiments for flux analysis during the leucine-limited fed-batch production phase in a 300-L bioreactor. Based on two-dimensional (2D) nuclear magnetic resonance (NMR) measurements of (13)C-labeling patterns of cytoplasmic free metabolites, metabolic flux distributions in the central metabolism were successfully determined. Focusing on the highly concentrated metabolite L-glutamate, the working hypothesis was validated that the equilibration of labeling patterns in intracellular pools was much faster (up to 9.45 min) than the labeling period (3 h) used in the experiments. Analysis of anaplerotic reactions revealed that highly selective lysine production was accompanied by a significant reduction of decarboxylating reactions from 10 mol% to only 2 mol%, whereas PEP/pyruvate-carboxylating fluxes remained constant at about 40 mol% of consumed glucose. These results support the conclusion that an optimized C. glutamicum L-lysine producer should possess increased PEP carboxylase and/or pyruvate carboxylase activity combined with downregulated, decarboxylating fluxes consuming oxaloacetate/malate. The findings also illustrate the usefulness of the sensor reactor approach in the study of industrial fermentations.

Production process monitoring by serial mapping of microbial carbon flux distributions using a novel sensor reactor approach: I--Sensor reactor system.

A novel Sensor Reactor technology is presented which permits 13C labeling experiments for metabolic flux analysis during large-scale, semi-industrial, (fed-) batch fermentation processes deriving a series of flux maps that document fermentation courses in detail. The small-scale Sensor Reactor can be inoculated within 1.50-1.20s via a special inoculation unit with an inoculation volume accuracy of 1.025+/-0.021 L. The large-scale production reactor (here: 300 L) and the Sensor Reactor were run in parallel master/slave modes to control the current pH, temperature, pressure and dissolved oxygen values as changing set points for the Sensor Reactor. Using an automated pulsing technology, glucose pulses of 5 g/L could be realized within 0.51 s. The similarity of fermentations in the Sensor Reactor with the production process was demonstrated by studying L-lysine production with C. glutamicum during multiple, 'simulated' labeling experiments each lasting 2.5h. 'Real' labeling experiments are presented in Part II.

Production process monitoring by serial mapping of microbial carbon flux distributions using a novel Sensor Reactor approach: II--(13)C-labeling-based metabolic flux analysis and L-lysine production.

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 13C-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 (El 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 13C-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 13C-isotope labelings resulting in a powerful tool for quantitative pathway analysis during a batch fermentation. As a first application, 13C-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 13C-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.