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

Date

2010-02-01

Author

Celik, Eda
Çalık, Pınar
Oliver, Stephen G.

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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.

Subject Keywords

Biotechnology, Applied Microbiology and Biotechnology, Bioengineering

URI

https://hdl.handle.net/11511/37247

Journal

BIOTECHNOLOGY AND BIOENGINEERING

DOI

https://doi.org/10.1002/bit.22543

Collections

Department of Chemical Engineering, Article

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Citation Formats

E. Celik, P. Çalık, and S. G. Oliver, “Metabolic Flux Analysis for Recombinant Protein Production by Pichia pastoris Using Dual Carbon Sources: Effects of Methanol Feeding Rate,” BIOTECHNOLOGY AND BIOENGINEERING, pp. 317–329, 2010, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/37247.