Engineering CHO cells for improved central carbon and energy metabolism
© Wilkens and Gerdtzen; licensee BioMed Central Ltd. 2011
Published: 22 November 2011
Investigations have shown animal cell cultures’ performance, in terms of cell proliferation and production of recombinant protein, are negatively affected by both lactate's concentration and its specific production rate. In a previous work, we determined that lactate production was caused by pyruvate accumulation due to its high synthesis rate in the glycolitic pathway and limited consumption in the TCA cycle, which leads to lactate production . In this work, we use the ΔL/ΔHexose ratio in order to characterize the cells metabolic state. This ratio describes the lactate production rate vs. hexose consumption. Low ΔL/ΔHexose ratios indicate efficient metabolic states where carbons consumed are mainly used to support cell growth, protein synthesis or energy metabolism.
Cell engineering has been previously used to improve cultures’ performance by changing the expression of genes involved in metabolism and apoptosis, focusing on the modification of only one gene at the time. These works showed that after overexpression of genes such as fructose transporter (Slc2a5) and yeast’s pyruvate carboxylase (PYC) cells are able to achieve higher cell densities and lower lactate production than wild-type cells under the same culture conditions [2–4].
In this work we aim at introducing multiple changes in the cells’ genome in order to obtain an engineered cell line with reduced lactate production and enhanced energy metabolism, which is capable of achieving higher cell densities and with a longer lifespan. We propose to control both, carbon uptake and its use by the TCA cycle. Cells were transfected with the fructose transporter gene (Slc2a5) and pyruvate carboxylase gene (PYC). Metabolic flux redistribution was studied through metabolic flux analysis, comparing engineered cells and wild-type under normal culture conditions.
Materials and methods
Parameters for cell growth and ΔL/ΔHexose
µmax [10-2 hrs-1]
ΔL/ΔHexose values reached by the cultures are given in Table 1. CHO cells grown in high glucose show an inefficient metabolic state where most carbons consumed go towards lactate production. Engineered cells grown in glucose have lower lactate production per carbon consumed than wild-type cells (Figure 1.(b)).
Engineered cells show a better use of glucose, producing less lactate per glucose consumed, as reflected in their lower ΔL/ΔHexose. In addition, CHO-PYC cells are able to produce less lactate and achieve a longer lifespan than wild-type cells. CHO-Slc2a5 cells have higher fructose uptake rates than FC and are able to achieve longer lifespans and higher cell densities.
CHO-PYC-Slc2a5 cells have the highest µmax among all experiments yet they produce more lactate than FC. Most of the lactate is produced in the lag phase. The fact that cells are capable of growing in fructose as well as in glucose and have a better ΔL/ΔHexose than GC, indicates that there is room for further improvement of this system.
Metabolic flux analysis
Figure 1.(c) shows the flux distribution of the different cultures for central carbon metabolism during mid exponential growth phase. CHO cells grown in fructose have lower amounts of carbon directed towards energy metabolism. Both CHO-Slc2a5 and CHO-PYC-Slc2a5 have higher fluxes in glycolysis and TCA cycle than FC, consistent with higher cell density. CHO-PYC cells consume lower amounts of glucose than GC, and most of it is directed towards the TCA cycle. CHO-Slc2a5 cells consume higher amounts of fructose than FC and most of it is directed towards the TCA cycle. CHO-PYC-Slc2a5 cells show a more active metabolism than FC, consuming more fructose, with higher TCA cycle fluxes and lactate production, while reaching higher cell densities than the control.
It is possible to modify cells for a more efficient metabolism in media supplemented with glucose and fructose using cell engineering. Engineered cells show enhanced viability and more efficient metabolic states under high glucose or fructose concentrations than the controls.
We would like to thank Dr. Roland Wagner for the PCMVSHE-PYC2 vector, Dr. Mariella Bollati for the pcDNA3.1(+)zeo vector and the Genetically Engineered Mouse Facility at M.D. Anderson Cancer Center for the Hygromycine resistance cassette. This work was supported by FONDECYT Initiation Grant 11090268.
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