title: Expression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells.
abstract: Recently, a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to reconstitute the missing link between glycolysis and TCA, thus increasing the flux of glucose into the TCA and resulting in a higher intracellular ATP content. Now, these metabolically engineered cells have been additionally transfected with a plasmid bearing the gene for human erythropoietin. EPO yield and substrate-specific productivity of the recombinant BHK-21 cells have been compared to control cells without the PYC2-gene but transfected with the plasmid coding for the expression of the selection genes and EPO. PYC2-expressing clones showed a 2-fold higher glucose-specific productivity and a 2-fold higher product concentration in a continuously perfused bioreactor. Moreover, the PYC2 expression enabled the cells to become more resistant to low glucose concentrations in the culture medium. They could produce at nearly maximum productivity under glucose-limiting conditions of 0.05-1 gl(-1) that guaranteed a reduced accumulation of lactate in fed-batch production systems. Due to the fact that PYC2-expressing cells are characterized by reduced glucose consumption, a prolonged production phase in bioreactors can be maintained. Based on the demand not to fall short of 80% cell viability for the production, EPO could be produced for 2 days (30%) longer compared to the control due to a more economic exploitation of glucose, and the prolonged viability period of the cells using a batch cultivation driven by glutamine limitation.
authors.affiliation: Department of Cell Culture Technology (ZKT), National Research Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
authors.forename: Noushin
authors.initials: N
authors.lastname: Irani
authors.forename: Alejandro José
authors.initials: AJ
authors.lastname: Beccaria
authors.forename: Roland
authors.initials: R
authors.lastname: Wagner
mesh_terms: D000818:Animals; D019149:Bioreactors; D002460:Cell Line; D006224:Cricetinae; D003470:Culture Media; D003593:Cytoplasm / Q000201:enzymology; D004274:DNA, Recombinant / Q000096:biosynthesis; D004921:Erythropoietin / Q000138:chemical synthesis* / Q000235:genetics*; D005822:Genetic Vectors / Q000096:biosynthesis; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D011766:Pyruvate Carboxylase / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics*; D014162:Transfection
chemical_list: D003470:Culture Media; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D005973:Glutamine; D004921:Erythropoietin; D011766:Pyruvate Carboxylase