Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate.
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 micro(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.
Department of Chemical Engineering, Middle East Technical University, Ankara, Turkey.
Eda
E
Celik
Pinar
P
Calik
Stephen G
SG
Oliver
D002244:Carbon / Q000378:metabolism*; D004921:Erythropoietin / Q000378:metabolism*; D006801:Humans; D007218:Industrial Microbiology; D000432:Methanol / Q000378:metabolism*; D008954:Models, Biological; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D013012:Sorbitol / Q000378:metabolism
D011994:Recombinant Proteins; D004921:Erythropoietin; D013012:Sorbitol; D002244:Carbon; D000432:Methanol