(623q) Engineering Galactose Metabolism for Improved Cell Growth of CHO Cells In Culture

The use of alternative carbon sources have been studied for the reduction of lactate in mammalian cell culture, as they are metabolized more slowly than glucose. This reduction in carbon uptake leads to lower production of metabolic end-products. Unfortunately, cultures grown in fructose and galactose are unable to achieve high cell growth rates, so they are not used as sole carbon sources. In a previous work we studied cultures in a mixture of glucose and galactose, and observed that glycolitic fluxes during glucose consumption were very high, causing pyruvate accumulation. Increased lactate production was linked to this high flux of pyruvate production [1]. During galactose consumption the flux towards pyruvate production was decreased compared to the culture in glucose, causing a metabolic shift towards lactate consumption to supply pyruvate for energy production.

A previous work showed that when CHO cells were transfected with a fructose transporter they were able to achieve higher cell densities than the wild type [2]. Other studies have observed that overexpression of limiting step enzymes lead to increased flux in the metabolic pathways associated [3,4]. For instance, overexpression of the PYC gene and MDH II enhanced energy metabolism enabling higher fluxes through the TCA cycle.

In this work we propose to improve the metabolism of CHO cells growing in galactose by introducing multiple changes in key steps of galactose metabolism. We determined that the limiting steps of galactose metabolism were transport and phosphorilation of galactose by galactokinase. A pIRES vector expressing neo resistance gene was constructed for expression of the GALK1 and GLUT8 genes named pIRES-GalMet. Expression of the GLUT 8 gene the transporter can be found in the lysosome membrane, but by changing the [E]XXX[LL] motif to [E]XXX[AA] the localization changes to the cytoplasmatic membrane [5]. A CHO TF 70R producing tPA cell line was transfected with the pIRES-GalMet vector and the best clone was selected by limit dilution in media containing geneticine. Cells were grown in galactose and combined glucowse and galactose. Cell growth improved significantly with respect to the control, with reduced lactate production. As a result we observe an extension on the culture´s lifespan and increased protein synthesis.

This strategy of cell engineering, were multiple changes in key points of the metabolism are targeted, enables cells to grow in alternative carbon sources such as galactose. The engineered cells have the ability to achieve higher cell growth rate, maintain high density cultures, extendend lifespan and higher recombinant yield.

[1] Wilkens, C.A., Altamirano, C., Gerdtzen, Z.P., Biotechnol. Bioprocess Eng (2011, accepted)

[2] Wlaschin, K.F., Hu, W-S., J Biotechnol, 131(2):168-76 (2007)

[3] Irani, N., Wirth, M., van Den Heuvel, J., Wagner, R., Biotechnol Bioeng, 66(4):238-46 (1999)

[4] Chong, W.P., Reddy, S.G., Yusufi, F.N., Lee, D.Y., Wong, N.S., Heng, C.K., Yap, M.G., Ho, Y.S., J Biotechnol, 147(2):116-21 (2010)

[5] Diril M.K., Schmidt S. Krauß M., Gawlik V., Joost H-G., Schürmann A., Haucke V., Augustin R., FEBS Journal 276: 3729–3743(2009)