Mulukutla Bhanu Chandra, Yongky Andrew, Grimm Simon, Daoutidis Prodromos, Hu Wei-Shou
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, United States of America.
PLoS One. 2015 Mar 25;10(3):e0121561. doi: 10.1371/journal.pone.0121561. eCollection 2015.
Cultured mammalian cells exhibit elevated glycolysis flux and high lactate production. In the industrial bioprocesses for biotherapeutic protein production, glucose is supplemented to the culture medium to sustain continued cell growth resulting in the accumulation of lactate to high levels. In such fed-batch cultures, sometimes a metabolic shift from a state of high glycolysis flux and high lactate production to a state of low glycolysis flux and low lactate production or even lactate consumption is observed. While in other cases with very similar culture conditions, the same cell line and medium, cells continue to produce lactate. A metabolic shift to lactate consumption has been correlated to the productivity of the process. Cultures that exhibited the metabolic shift to lactate consumption had higher titers than those which didn't. However, the cues that trigger the metabolic shift to lactate consumption state (or low lactate production state) are yet to be identified. Metabolic control of cells is tightly linked to growth control through signaling pathways such as the AKT pathway. We have previously shown that the glycolysis of proliferating cells can exhibit bistability with well-segregated high flux and low flux states. Low lactate production (or lactate consumption) is possible only at a low glycolysis flux state. In this study, we use mathematical modeling to demonstrate that lactate inhibition together with AKT regulation on glycolysis enzymes can profoundly influence the bistable behavior, resulting in a complex steady-state topology. The transition from the high flux state to the low flux state can only occur in certain regions of the steady state topology, and therefore the metabolic fate of the cells depends on their metabolic trajectory encountering the region that allows such a metabolic state switch. Insights from such switch behavior present us with new means to control the metabolism of mammalian cells in fed-batch cultures.
培养的哺乳动物细胞表现出较高的糖酵解通量和较高的乳酸产量。在用于生物治疗性蛋白质生产的工业生物过程中，向培养基中补充葡萄糖以维持细胞的持续生长，导致乳酸积累到高水平。在这种补料分批培养中，有时会观察到代谢从高糖酵解通量和高乳酸产量状态转变为低糖酵解通量和低乳酸产量甚至乳酸消耗状态。而在其他具有非常相似培养条件、相同细胞系和培养基的情况下，细胞继续产生乳酸。向乳酸消耗的代谢转变与该过程的生产力相关。表现出向乳酸消耗代谢转变的培养物的滴度高于未发生转变的培养物。然而，触发向乳酸消耗状态（或低乳酸产生状态）代谢转变的线索尚未确定。细胞的代谢控制通过诸如AKT途径等信号通路与生长控制紧密相连。我们之前已经表明，增殖细胞的糖酵解可以表现出双稳态，具有明显分离的高通量和低通量状态。只有在低糖酵解通量状态下才可能出现低乳酸产生（或乳酸消耗）。在本研究中，我们使用数学建模来证明乳酸抑制以及AKT对糖酵解酶的调节可以深刻影响双稳态行为，从而导致复杂的稳态拓扑结构。从高通量状态到低通量状态的转变只能在稳态拓扑结构的某些区域发生，因此细胞的代谢命运取决于其代谢轨迹是否遇到允许这种代谢状态转换的区域。这种转换行为的见解为我们提供了控制补料分批培养中哺乳动物细胞代谢的新方法。
