TY - JOUR

T1 - Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation

AU - Nicolae, Averina

AU - Wahrheit, Judith

AU - Bahnemann, Janina

AU - Zeng, An Ping

AU - Heinzle, Elmar

N1 - Funding information: We thank the Institute of Cell Culture Technology (University Bielefeld, Germany) for supplying the CHO K1 cells and the BMBF (German Federal Ministry of Education and Research) project SysCompart (project ID 031555D), part of the Systems Biology program, New Methods in Systems Biology (SysTec), for funding.

PY - 2014/4/28

Y1 - 2014/4/28

N2 - Background: Mapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation.Results: Intracellular fluxes of the CHO-K1 cell line central carbon metabolism were successfully determined for a complex network using non-stationary 13C metabolic flux analysis. Mass isotopomers of extracellular metabolites were determined using [U-13C6] glucose as labeled substrate. Metabolic compartmentation and extracellular transport reversibility proved essential to successfully reproduce the dynamics of the labeling patterns. Alanine and pyruvate reversibility changed dynamically even if their net production fluxes remained constant. Cataplerotic fluxes of cytosolic phosphoenolpyruvate carboxykinase and mitochondrial malic enzyme and pyruvate carboxylase were successfully determined. Glycolytic pyruvate channeling to lactate was modeled by including a separate pyruvate pool. In the exponential growth phase, alanine, glycine and glutamate were excreted, and glutamine, aspartate, asparagine and serine were taken up; however, all these amino acids except asparagine were exchanged reversibly with the media. High fluxes were determined in the pentose phosphate pathway and the TCA cycle. The latter was fueled mainly by glucose but also by amino acid catabolism.Conclusions: The CHO-K1 central metabolism in controlled batch culture proves to be robust. It has the main purpose to ensure fast growth on a mixture of substrates and also to mitigate oxidative stress. It achieves this by using compartmentation to control NADPH and NADH availability and by simultaneous synthesis and catabolism of amino acids.

AB - Background: Mapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation.Results: Intracellular fluxes of the CHO-K1 cell line central carbon metabolism were successfully determined for a complex network using non-stationary 13C metabolic flux analysis. Mass isotopomers of extracellular metabolites were determined using [U-13C6] glucose as labeled substrate. Metabolic compartmentation and extracellular transport reversibility proved essential to successfully reproduce the dynamics of the labeling patterns. Alanine and pyruvate reversibility changed dynamically even if their net production fluxes remained constant. Cataplerotic fluxes of cytosolic phosphoenolpyruvate carboxykinase and mitochondrial malic enzyme and pyruvate carboxylase were successfully determined. Glycolytic pyruvate channeling to lactate was modeled by including a separate pyruvate pool. In the exponential growth phase, alanine, glycine and glutamate were excreted, and glutamine, aspartate, asparagine and serine were taken up; however, all these amino acids except asparagine were exchanged reversibly with the media. High fluxes were determined in the pentose phosphate pathway and the TCA cycle. The latter was fueled mainly by glucose but also by amino acid catabolism.Conclusions: The CHO-K1 central metabolism in controlled batch culture proves to be robust. It has the main purpose to ensure fast growth on a mixture of substrates and also to mitigate oxidative stress. It achieves this by using compartmentation to control NADPH and NADH availability and by simultaneous synthesis and catabolism of amino acids.

KW - Chinese hamster ovary cells

KW - CHO

KW - Compartmentation

KW - Mammalian cell culture

KW - Mammalian metabolism

KW - Metabolic flux analysis

KW - Metabolic transport

KW - Mitochondria

KW - Reversibility

UR - http://www.scopus.com/inward/record.url?scp=84899952895&partnerID=8YFLogxK

U2 - 10.1186/1752-0509-8-50

DO - 10.1186/1752-0509-8-50

M3 - Article

C2 - 24773761

AN - SCOPUS:84899952895

VL - 8

JO - BMC Systems Biology

JF - BMC Systems Biology

SN - 1752-0509

IS - 1

M1 - 50

ER -