Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [13C, 1H] COSY NMR spectroscopy

Goudar, Chetan T.; Biener, Richard; Boisart, C.; Heidemann, Rüdiger; Piret, James M.; Graaf, Albert A. de; Konstantinov, Konstantin

doi:10.1016/j.ymben.2009.10.007

Cited by 97 publications

(83 citation statements)

References 46 publications

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“…About 40% of glucose was metabolized via oxPPP (remaining 60% via glycolysis), and 10% of pyruvate was converted to oxaloacetate by PC (remaining 90% was converted to acetyl-CoA by PDH). Goudar et al [114] demonstrated that results obtained using classical MFA only agreed well with 13 C-MFA results when the fluxes of oxPPP and PC reactions were fixed at the values obtained from 13 C-MFA analysis. Sengupta et al [115] also used a mixture of [1-13 C]glucose and [U-13 C]glucose tracers and measured 13 C-labeling in four metabolites of the pentose-phosphate pathway using LC-MS. Sengupta et al estimated oxPPP fluxes using 13 C-MFA in CHO cell cultures at the late stationary phase.…”

Section: Applications Of 13 C-tracers For Flux Analysis In Cho Cellsmentioning

confidence: 91%

Towards dynamic metabolic flux analysis in CHO cell cultures

Ahn

Antoniewicz

2011

Biotechnology Journal

111

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Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance.

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Section: Applications Of 13 C-tracers For Flux Analysis In Cho Cellsmentioning

confidence: 91%

Towards dynamic metabolic flux analysis in CHO cell cultures

Ahn

Antoniewicz

2011

Biotechnology Journal

111

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“…These results underscore the obvious and growing need for understanding the central metabolism of SYK-6 in association with lignin degradation to establish a robust molecular and cellular biology toolbox for further manipulation and engineering of the organism for lignin valorization. 13 C metabolic flux analysis ( 13 C-MFA) is a powerful technique for determining steady-state in vivo fluxes of central metabolic pathways in living cells (26)(27)(28)(29). 13 C-MFA also has been conducted to understand the metabolism of biofuel-and biochemical-producing microbial strains (30)(31)(32).…”

Section: Significancementioning

confidence: 99%

Decoding how a soil bacterium extracts building blocks and metabolic energy from ligninolysis provides road map for lignin valorization

Varman

Follenfant

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Sphingobium sp. SYK-6 is a soil bacterium boasting a well-studied ligninolytic pathway and the potential for development into a microbial chassis for lignin valorization. An improved understanding of its metabolism will help researchers in the engineering of SYK-6 for the production of value-added chemicals through lignin valorization. We used 13 C-fingerprinting, 13C metabolic flux analysis ( 13 C-MFA), and RNA-sequencing differential expression analysis to uncover the following metabolic traits: (i) SYK-6 prefers alkaline conditions, making it an efficient host for the consolidated bioprocessing of lignin, and it also lacks the ability to metabolize sugars or organic acids; (ii) the CO 2 release (i.e., carbon loss) from the ligninolysis-based metabolism of SYK-6 is significantly greater than the CO 2 release from the sugar-based metabolism of Escherichia coli; (iii) the vanillin catabolic pathway (which is the converging point of majority of the lignin catabolic pathways) is coupled with the tetrahydrofolate-dependent C1 pathway that is essential for the biosynthesis of serine, histidine, and methionine; (iv) catabolic end products of lignin (pyruvate and oxaloacetate) must enter the tricarboxylic acid (TCA) cycle first and then use phosphoenolpyruvate carboxykinase to initiate gluconeogenesis; and (v) 13 C-MFA together with RNA-sequencing differential expression analysis establishes the vanillin catabolic pathway as the major contributor of NAD(P)H synthesis. Therefore, the vanillin catabolic pathway is essential for SYK-6 to obtain sufficient reducing equivalents for its healthy growth; cosubstrate experiments support this finding. This unique energy feature of SYK-6 is particularly interesting because most heterotrophs rely on the transhydrogenase, the TCA cycle, and the oxidative pentose phosphate pathway to obtain NADPH.

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“…Cytosolic G6P and 3PG concentrations decreased during the cultivation which correlated with the glutamine and glucose availability. Multiple metabolic flux studies [49][50][51] had already shown that intracellular flux distributions severely depend on the availability of glutamine and the growth phase [52,53]. While high glutamine consumption fuels lactate and pyruvate formation, the flux scenario changes completely as soon as glutamine is depleted.…”

Section: The Influence Of Growth Phase and Substrate Availability On mentioning

confidence: 99%

Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

Matuszczyk

Teleki

Pfizenmaier

2015

Biotechnology Journal

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Mammalian cells show a compartmented metabolism. Getting access to subcellular metabolite pools is of high interest to understand the cells' metabolomic state. Therefore a protocol is developed and applied for monitoring compartment-specific metabolite and nucleotide pool sizes in Chinese hamster ovary (CHO) cells. The approach consists of a subtracting filtering method separating cytosolic components from physically intact mitochondrial compartments. The internal standards glucose-6-phosphate and cis-aconitate were chosen to quantify cytosolic secession and mitochondrial membrane integrity. Extracts of related fractions were studied by liquid chromatography-isotope dilution mass spectrometry for the absolute quantification of a subset of glycolytic and tricarboxylic acid cycle intermediates together with the adenylate nucleotides ATP, ADP and AMP. The application of the protocol revealed highly dynamic changes in the related pool sizes as a function of distinct cultivation periods of IgG1 producing CHO cells. Mitochondrial and cytosolic pool dynamics were in agreement with anticipated metabolite pools of independent studies. The analysis of adenosine phosphate levels unraveled significantly higher ATP levels in the cytosol leading to the hypothesis that mitochondria predominantly serve for fueling ATP into the cytosol where it is tightly controlled at physiological adenylate energy charges about 0.9.

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Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [13C, 1H] COSY NMR spectroscopy

Cited by 97 publications

References 46 publications

Towards dynamic metabolic flux analysis in CHO cell cultures

Towards dynamic metabolic flux analysis in CHO cell cultures

Decoding how a soil bacterium extracts building blocks and metabolic energy from ligninolysis provides road map for lignin valorization

Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

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