Production process monitoring by serial mapping of microbial carbon flux distributions using a novel Sensor Reactor approach: II—13C-labeling-based metabolic flux analysis and l-lysine production
Introduction
Amino acid production plays an important role in the fermentation industry. In particular, the bacterium Corynebacterium glutamicum and its closely related species C. glutamicum ssp. flavum and lactofermentum are widely used for the commercial production of lysine and glutamate. The key metabolic reactions have been intensively studied in coryneform bacteria and these organisms have a glycolytic pathway, a pentose phosphate pathway (PPP), a tricarboxylic acid (TCA) cycle and a glyoxylate bypass (Kinoshita, 1985). For the synthesis of lysine and other amino acids of the aspartate family, anaplerotic reactions are of key importance as they supply oxaloacetate, a direct precursor of aspartate. In C. glutamicum, the anaplerotic synthesis of oxaloacetate via carboxylation of C3 metabolites is catalyzed by phosphoenolpyruvate (PEP) carboxylase and pyruvate carboxylase (Peters-Wendisch, Eikmanns, Thierbach, Bachmann, & Sahm (1993), Peters-Wendisch, Wendisch, Paul, Eikmanns, & Sahm (1997)). In addition to having two C3-carboxylating enzymes, C. glutamicum also possesses the C4-decarboxylating enzymes PEP carboxykinase, oxaloacetate decarboxylase and malic enzyme during growth on glucose (Jetten and Sinskey (1993), Jetten and Sinskey (1995); Cocaign-Bousquet et al., 1996). Previous work on 13C-labeling-based metabolic flux analysis in this organism demonstrated that both C3-carboxylation and C4-decarboxylation occur simultaneously in vivo (Sonntag et al., 1995; Marx, Eikmanns, Sahm, de Graaf, & Eggeling (1999), Marx, Striegel, de Graaf, Sahm, & Eggeling (1997)). Petersen et al. (2000) were subsequently able to quantify the individual fluxes at the anaplerotic node and showed that the PEP carboxykinase is the enzyme responsible for recycling two-thirds of the anaplerotically synthesized oxaloacetate to PEP.
Industrial amino acid production uses mostly very large-scale (fed-) batch fermentations. In contrast, 13C-based metabolic flux analyses of C. glutamicum have been primarily performed with cells growing in carbon-limited laboratory chemostat cultures (Marx et al., 1996; Marx, Striegel, de Graaf, Sahm, & Eggeling (1997), Marx, Eikmanns, Sahm, de Graaf, & Eggeling (1999); Petersen et al. (2000), Petersen et al. (2001)) or with cells in small-scale shake-flask batch fermentations (Sonntag et al., 1995; Wendisch et al., 2000) and fed-batch processes using stoichiometrically based mass balances (Vallino and Stephanopoulos, 1993). No 13C-based data about the intracellular fluxes in C. glutamicum during lysine production in large-scale semi-industrial cultivations are so far available. Therefore, to bring 13C-based flux analysis up to the industrial level, we combined for the first time the Sensor Reactor approach described by El Massaoudi et al. (Metab. Eng., submitted) with nuclear magnetic resonance (NMR) analysis, metabolite balancing methods and the mathematical analysis of 13C-isotope labeling to obtain a series of flux maps documenting the changes of intracellular flux distributions during a typical pilot-size batch fermentation process.
In the first application of the technique we investigated the central metabolic fluxes in leucine auxotrophic, lysine-producing C. glutamicum MH20-22B in a 300-L fermenter, with a particular focus on the variation of the flux distribution at the anaplerotic node with increasing lysine synthesis.
Section snippets
Bacterial organism, cultivation and 13C labeling
C. glutamicum MH20-22B, derived from C. glutamicum ATCC 13032 (wild-type) by undirected mutagenesis, was used as a lysine-producing strain (Schrumpf, Eggeling, Sahm (1992), Schrumpf et al. (1991)). For large-scale batch conditions, cultivation was carried out in a production reactor (total reactor volume 300 L) which was connected to a small Sensor Reactor (1 L). The detailed principles of the approach as well as the medium compositions used are described by El Massaoudi et al. (Metab. Eng.,
Comparison of the growth of C. glutamicum MH20-22B and extracellular metabolite concentrations in production and Sensor Reactor
Using the Sensor Reactor technology, a batch experiment with lysine-producing C. glutamicum MH20-22B was carried out to investigate the alterations of the in vivo metabolic flux distribution during lysine production. Therefore, during exponential growth in the production reactor, with a growth rate of 0.20±0.01 h−1, a series of three parallel fermentations was performed during three consecutive periods (Fig. 1). For the estimation of the concentration courses of the main carbon source glucose,
Acknowledgements
We thank Prof. W. Wiechert (University of Siegen, Germany) for supplying the mathematical tools for the flux analysis. A. Drysch received a scholarship from the Deutsche Forschungsgemeinschaft's graduate student training program in molecular physiology (University of Düsseldorf, Germany).
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