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

Abstract

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 C₄-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.

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.