New manuscript accepted
The influence of cell growth and enzyme activity changes on intracellular metabolite dynamics in AGE1.HN.AAT cells
Rath AG, Rehberg M, Janke R, Genzel Y, Scholz S, Noll T, Rose T, Sandig V, Reichl U
Journal of Biotechnology, accepted
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-depths understanding 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 exponential, transient and stationary growth phases 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 20 L batch cultivations with AGE1.HN.AAT cells. Our results, obtained by modeling the cell growth and consumption of main substrates for the different growth phases, showed a strong correlation of the dynamics of intracellular metabolite pools with specific uptake rates of glucose and glutamine. Furthermore, cross correlation analysis confirmed a regulation of phosphofructokinase and pyruvate kinase during the transient growth phase. As expected, hierarchical cluster analysis showed a clear separation into an upper and lower part of glycolysis. By analyzing maximum in vitro enzyme activities we found low activities of pyruvate dehydrogenase and pyruvate carboxylase for metabolite transfer into the citric acid cycle resulting in high lactate release (Warburg effect). As maximum in vitro enzyme activities remain mainly unchanged during the time course of both cultivations, we hypothesize that apart from their regulation by substrates and products, key enzymes of glycolysis in AGE1.HN.AAT cells are strongly modulated by allosteric regulators. 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 suggest how the metabolic networks of immortalized mammalian cells are regulated by changes of enzyme levels and metabolite pools over the time course of batch cultivations. Eventually, it enables the use of cell engineering strategies, which can improve the availability of building blocks for biomass synthesis by increasing glucose as well as glutamine fluxes, and the knockdown of the glutamine synthetase to prevent unnecessary dissipation of ATP, to yield a cell line with optimized growth characteristics and increased overall productivity.