Process-Orientated Metabolic Engineering: Cell Lines with New Properties in Nutrient Exploitation and Protein Glycosylation

Abstract

Although well accepted as reliable and safe production hosts for manufacturing pharmaglyco-proteins, continuous mammalian cell lines, to a large extent, have the metabolic disadvantages of being unable to completely oxidize glucose and to secrete precisely processed human-type glycoproteins. Metabolic engineering has been shown to have a considerable impact on the improvement of CCLs. In general, three different major strategies can be distinguished. First, the generation of proliferation-controlled cells by introducing genes affecting cell growth. Second, modifications of the protein-processing machinery by introducing specific glycosyltransferases and third, strategies to reconstitute the cellular metabolism. The modification of the primary metabolism, however, is believed to be hard to manipulate because the primary metabolism network is very complex and rigid. Therefore, hardly any efforts towards a reconstitution and improvement of the primary metabolism by metabolic engineering have been undertaken in mammalian cell lines so far. Only strategies that activate channels for glycolytic metabolites to the TCA, resulting in a complete oxidation of glucose, or those, which reactivate the primary end products for energy production, can overcome the basic problems of CCLs. By introducing a low-KM hexokinase, the energy level of mammalian cell lines could be substantially elevated resulting in higher productivity. In addition, glucose carbons could be more efficiently channeled into the TCA by a yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells. This strategy enables the cells to transfer glycolysis-derived pyruvate into malate, which can enter the TCA cycle for complete oxidation.