Metabolic Flux Analysis for Recombinant Protein Production by Pichia pastoris Using Dual Carbon Sources: Effects of Methanol Feeding Rate

Celik E., ÇALIK P., Oliver S. G.

BIOTECHNOLOGY AND BIOENGINEERING, vol.105, no.2, pp.317-329, 2010 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 105 Issue: 2
  • Publication Date: 2010
  • Doi Number: 10.1002/bit.22543
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.317-329
  • Hacettepe University Affiliated: No


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 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 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 la shift in metabolism towards the dissimilatory pathway that corre- sponded 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 mu(0). 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. (C) 2009 Wiley Periodicals, Inc.