title: The influence of cell growth and enzyme activity changes on intracellular metabolite dynamics in AGE1.HN.AAT cells.
abstract: Optimization of bioprocesses with mammalian cells mainly concentrates on cell engineering, cell screening and medium optimization to achieve enhanced cell growth and productivity. For improving cell lines by cell engineering techniques, in-depth understandings of the regulation of metabolism and product formation as well as the resulting demand for the different medium components are needed. In this work, the relationship of cell specific growth and uptake rates and of changes in maximum in vitro enzyme activities with intracellular metabolite pools of glycolysis, pentose phosphate pathway, citric acid cycle and energy metabolism were determined for batch cultivations with AGE1.HN.AAT cells. Results obtained by modeling cell growth and consumption of main substrates showed that the dynamics of intracellular metabolite pools is primarily linked to the dynamics of specific glucose and glutamine uptake rates. By analyzing maximum in vitro enzyme activities we found low activities of pyruvate dehydrogenase and pyruvate carboxylase which suggest a reduced metabolite transfer into the citric acid cycle resulting in lactate release (Warburg effect). Moreover, an increase in the volumetric lactate production rate during the transition from exponential to stationary growth together with a transient accumulation of fructose 1,6-bisphosphate, fructose 1-phosphate and ribose 5-phosphate point toward an upregulation of PK via FBP. Glutaminase activity was about 44-fold lower than activity of glutamine synthetase. This seemed to be sufficient for the supply of intermediates for biosynthesis but might lead to unnecessary dissipation of ATP. Taken together, our results elucidate regulation of metabolic networks of immortalized mammalian cells by changes of metabolite pools over the time course of batch cultivations. Eventually, it enables the use of cell engineering strategies to improve the availability of building blocks for biomass synthesis by increasing glucose as well as glutamine fluxes. An additional knockdown of the glutamine synthetase might help to prevent unnecessary dissipation of ATP, to yield a cell line with optimized growth characteristics and increased overall productivity.
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering Group, Sandtorstr. 1, 39106 Magdeburg, Germany. Electronic address: rath@mpi-magdeburg.mpg.de.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Rath
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering Group, Sandtorstr. 1, 39106 Magdeburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Rehberg
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering Group, Sandtorstr. 1, 39106 Magdeburg, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Janke
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering Group, Sandtorstr. 1, 39106 Magdeburg, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Genzel
authors.affiliation: Institute of Cell Culture Technology, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Scholz
authors.affiliation: Institute of Cell Culture Technology, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Noll
authors.affiliation: ProBioGen AG, Goethestr. 54, 13086 Berlin, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rose
authors.affiliation: ProBioGen AG, Goethestr. 54, 13086 Berlin, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Sandig
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering Group, Sandtorstr. 1, 39106 Magdeburg, Germany; Chair for Bioprocess Engineering, Otto-von-Guericke-University, Universitätsplatz 2, 39106 Magdeburg, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Reichl
mesh_terms: D019149:Bioreactors; D001709:Biotechnology; D060846:Cell Engineering; D002460:Cell Line / Q000166:cytology / Q000378:metabolism; D049109:Cell Proliferation / Q000502:physiology*; D002470:Cell Survival; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000502:physiology; D011770:Pyruvate Kinase / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; C043392:SERPINA1 protein, human; D000515:alpha 1-Antitrypsin; D011770:Pyruvate Kinase
keywords: Central carbon metabolism; Enzyme activity; Intracellular metabolite; Mammalian cell; Mathematical model; Recombinant protein