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

Celik, Eda
Çalık, Pınar
Oliver, Stephen G.
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.