Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate

Authors

  • Eda Çelik,

    1. Department of Chemical Engineering, Industrial Biotechnology and Metabolic Engineering Laboratory, Middle East Technical University, 06531 Ankara; telephone: 90-312-210-43-85; fax: 90-312-210-26-00, Turkey
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  • Pınar Çalık,

    Corresponding author
    1. Department of Chemical Engineering, Industrial Biotechnology and Metabolic Engineering Laboratory, Middle East Technical University, 06531 Ankara; telephone: 90-312-210-43-85; fax: 90-312-210-26-00, Turkey
    • Department of Chemical Engineering, Industrial Biotechnology and Metabolic Engineering Laboratory, Middle East Technical University, 06531 Ankara; telephone: 90-312-210-43-85; fax: 90-312-210-26-00, Turkey.
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  • Stephen G. Oliver

    1. Department of Biochemistry, University of Cambridge, Cambridge, UK
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Abstract

The intracellular metabolic fluxes through the central carbon pathways in the bioprocess for recombinant human erythropoietin (rHuEPO) production by Pichia pastoris (Mut+) were calculated to investigate the metabolic effects of dual carbon sources (methanol/sorbitol) and the methanol feed rate, and to obtain a deeper understanding of the regulatory circuitry of P. pastoris, using the established stoichiometry-based model containing 102 metabolites and 141 reaction fluxes. Four fed-batch operations with (MS-) and without (M-) sorbitol were performed at three different constant specific growth rates (h−1), and denoted as M-0.03, MS-0.02, MS-0.03, and MS-0.04. Considering the methanol consumption pathway, the M-0.03 and MS-0.02 conditions produced similar effects and had >85% of formaldehyde flux towards the assimilatory pathway. In contrast, the use of the dual carbon source condition generated a shift in metabolism towards the dissimilatory pathway that corresponded to the shift in dilution rate from MS-0.03 to MS-0.04, indicating that the methanol feed exceeded the metabolic requirements at the higher µ0. Comparing M-0.03 and MS-0.03 conditions, which had the same methanol feeding rates, sorbitol addition increased the rHuEPO synthetic flux 4.4-fold. The glycolysis, gluconeogenesis, and PPP pathways worked uninterruptedly only at MS-0.02 condition. PPP and TCA cycles worked with the highest disturbances at MS-0.04 condition, which shows the stress of increased feeding rates of methanol on cell metabolism. Biotechnol. Bioeng. 2010; 105: 317–329. © 2009 Wiley Periodicals, Inc.

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