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.


BOZOGLU, F; OZILGEN, M; BAKIR, U (Elsevier BV, 1987-09-01)
Survival kinetics of lactic acid starter cultures were modeled considering the microorganism and external medium interfacial area as the critical factors determining the resistance of the microorganisms to freeze-drying. Surviving fraction of the microorganisms increased with the increasing biomass concentration during freeze-drying, and this is attributed to the mutual shielding effect of the microorganisms against the severe conditions of the external medium. Survival of the microorganisms over the storag...
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Yilmaz, Ebru I.; Caydasi, Ayse K.; Özcengiz, Gülay (Springer Science and Business Media LLC, 2008-01-01)
The aspartate pathway of Streptomyces clavuligerus is an important primary metabolic pathway which provides substrates for beta-lactam synthesis. In this study, the hom gene which encodes homoserine dehydrogenase was cloned from the cephamycin C producer S. clavuligerus NRRL 3585 and characterized. The fully sequenced open reading frame encodes 433 amino acids with a deduced M (r) of 44.9 kDa. The gene was heterologously expressed in the auxotroph mutant Escherichia coli CGSC 5075 and the recombinant protei...
Kinetics of amino acid production by over-producer mutant microorganisms
Özilgen, Mustafa (Elsevier BV, 1988-2)
Experimental data were modeled based on the literature for the production of nine different amino acids by ten different mutant over-producer microorganisms. Mathematical modeling helped to better understand common aspects of these fermentations, which were not stressed previously by plotting the data only. A Luedeking-Piret type of a model was found to be valid for representing all the amino acid production data. This result indicated the similarity among the amino acid production patterns of all the mutan...
Kinetic analyses of the effects of temperature and light intensity on growth, hydrogenm production and organic acid utilization by rhodobacter capsulatus
Sevinç, Pelin; Gündüz, Ufuk; Department of Biotechnology (2010)
Effects of temperature and light intensity on photofermentative hydrogen production by Rhodobacter capsulatus DSM1710 by use of acetic and lactic acids as substrates were studied. Experiments were conducted at 20, 30 and 38oC incubator temperatures under light intensities in the 1500 – 7000 lux range. pH of the medium and quantity of hydrogen forming together with quantity of biomass, and concentrations of acetic, lactic, formic, butyric and propionic acids in the medium were determined periodically. Growth...
Homologous expression of aspartokinase (ask) gene in Streptomyces clavuligerus and its hom-deleted mutant: Effects on cephamycin C production
Özcengiz, Gülay; Ünsaldi, Eser; Taşkin, Bilgin; Liras, Paloma; Piret, Jacqueline (Informa UK Limited, 2010-05-01)
Abstract In this study, the effect of homologous multiple copies of the ask gene, which encodes aspartokinase catalyzing the first step of the aspartate pathway, on cephamycin C biosynthesis in S. clavuligerus NRRL 3585 and its hom mutant was investigated. The intracellular pool levels of aspartate pathway amino acids accorded well with the Ask activity levels in TB3585 and AK39. When compared with the control strain carrying vector alone without any gene insert, amplification of the ask gene in the w...
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: