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Improvement of the primary metabolism of cell cultures by introducing a new cytoplasmic pyruvate carboxylase reaction
Author(s) -
Irani Noushin,
Wirth Manfred,
van den Heuvel Joop,
Wagner Roland
Publication year - 1999
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(1999)66:4<238::aid-bit5>3.0.co;2-6
Subject(s) - citric acid cycle , glycolysis , pyruvate carboxylase , biochemistry , oxidative phosphorylation , metabolism , glutamine , flux (metallurgy) , adenosine triphosphate , cell culture , biology , chemistry , enzyme , amino acid , genetics , organic chemistry
Continuous mammalian cell lines are important hosts for the production of biological pharmaceuticals. However, these cell lines show some severe disorders in primary metabolism, which they have in common with many cancer cells. This leads to a high throughput of substrates giving a low energy yield and ample toxic side products such as lactate and ammonia. Because the enzymatic connection between glycolysis and the tricarboxylic acid cycle (TCA) is very poor, glucose is mainly degraded via oxidative glycolysis. It will be shown that introducing a pyruvate carboxylase gene expressed in the cytoplasma into a continuous BHK‐21 cell line, and thus reconstituting the missing link between glycolysis and TCA, can reduce this problem. Thus, glucose consumption could be reduced by a factor of four and glutamine utilization up to a factor of two, compared with control. Moreover, a 1.4‐fold‐higher adenosine triphosphate (ATP) content was achieved. The flux of labeled [ 14 C]‐glucose into the TCA is shown to be enhanced, indicating a higher rate of oxidative glucose degradation. Host cell lines with an improved energy metabolism will therefore result in better exploitation of substrates, an increasing yield by the more efficient use of carbon source, and higher product integrity combined with lower production costs. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 238–246, 1999.