title: Improving glucose and glutamine metabolism of human HEK 293 and Trichoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme.
abstract: Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect (Trichoplusia ni High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed.
authors.affiliation: Animal Cell Technology Dept., Biotechnology Research Institute, Montréal, Québec, Canada H4P 2R2, and Cell Culture Technology Dept., German Research Centre for Biotechnology mbH, Braunschweig, Germany.
authors.forename: Cynthia B
authors.initials: CB
authors.lastname: Elias
authors.forename: Eric
authors.initials: E
authors.lastname: Carpentier
authors.forename: Yves
authors.initials: Y
authors.lastname: Durocher
authors.forename: Louis
authors.initials: L
authors.lastname: Bisson
authors.forename: Roland
authors.initials: R
authors.lastname: Wagner
authors.forename: Amine
authors.initials: A
authors.lastname: Kamen
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D002452:Cell Count; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005818:Genetic Engineering / Q000379:methods*; D005947:Glucose / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology; D008660:Metabolism / Q000235:genetics / Q000502:physiology; D009036:Moths / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000096:biosynthesis / Q000235:genetics; D011766:Pyruvate Carboxylase / Q000096:biosynthesis* / Q000235:genetics; D011786:Quality Control; D014162:Transfection / Q000379:methods; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014764:Viral Proteins; D005973:Glutamine; D007652:Oxo-Acid-Lyases; C400770:Pyc protein, Streptomyces clavuligerus; D011766:Pyruvate Carboxylase; D005947:Glucose