Metabolic reaction network of Pichia pastoris with glycosylation reactions: Flux analysis for erythropoietin production

Abstract

BACKGROUND Biochemical reaction network of Pichia pastoris was improved by including N‐ glycosylation pathway reactions to determine the intracellular reaction rates for glycosylated protein production. RESULTS When co‐substrate sorbitol was replaced with mannitol, 12‐fold increase in erythropoietin flux was obtained as a result of 1.2‐ and 2.4‐fold increase in glucose‐6‐phosphate (G6P) formation from fructose‐6‐phosphate (F6P) and 3‐phospho‐D‐glycerate (3PG) formation from glyceraldehyde‐3‐phosphate (G3P), respectively. Thus, to analyse the fluxes around F6P and the fluxes of the glycosylation pathway reactions in depth, the biochemical reaction network of P. pastoris containing 204 reactions and 129 metabolites was developed and used. The flux analysis for glycosylated erythropoietin production with three glycan residues having 12 mannoses (EPO‐Man12) reveals that the fluxes from G3P and guanosine 5′‐diphosphate (GDP) to F6P synthesis were 1.6‐ and 3‐fold higher than that of the fluxes determined for erythropoietin production with no glycans (EPO), respectively. Furthermore, the results of theoretical data‐based overproduction capacity show that the fluxes of EPO‐Man12 and partially humanized EPO with three glycan residues having five mannoses (EPO‐Man5) were 3.6 and 3.8 µmol gDCW‐1 h‐1, respectively, when the methanol and mannitol uptake rates were 2.10 mmol gDCW‐1 h‐1 and 1.49 mmol gDCW‐1 h‐1. For glycoprotein production, fluxes around F6P were found to be important as UDP‐N‐acetyl‐α‐D‐glucosamine (UDP‐GlcNAc) and guanosine 5′‐diphosphate‐D‐Mannose (GDP‐Man) were produced from F6P. Thus the co‐substrate mannitol does not repress AOX promoter but is easily converted to F6P, enhancing glycoprotein production. CONCLUSION A metabolic reaction network for P. pastoris with glycosylation reactions is proposed for the determination of fluxes for glycoprotein production by P. pastoris. © 2013 Society of Chemical Industry

Genome‐scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins

“…A). This is in contrast to previous computational modelling that was performed of N‐glycosylation in P. pastoris (Eskitoros et al, ), where it was concluded that yields of humanized N‐glycosylated proteins are higher than of native N‐glycosylated proteins. The reduced theoretical yield of humanized in comparison to native N‐glycosylation can be explained by the size of the N‐glycan complex, which contains 70 and 84 carbons in native and humanized forms respectively.…”

“…In comparison with other models of N‐glycosylation in P. pastoris (Barrigon et al, ; Eskitoros et al, ), our model is in the context of the whole of P. pastoris metabolism, allows both native and humanized N‐glycosylation of multiple recombinant proteins and estimates N‐glycosylation of the native proteome.…”

“…The reduced theoretical yield of humanized in comparison to native N‐glycosylation can be explained by the size of the N‐glycan complex, which contains 70 and 84 carbons in native and humanized forms respectively. In comparison, the model of (Eskitoros et al, ) produces partially humanized N‐glycosylation, which requires lower amount of carbons.…”

“…One of the tools of metabolic engineering is the use of computational models that describe the metabolic pathways in cells and can aid in the identification of new targets or strategies for further metabolic engineering (Kerkhoven et al, ). A metabolic model describing N‐glycosylation in P. pastoris has previously been constructed (Eskitoros et al, ), however, this model only consisted of a subset of the metabolic reaction present in P. pastoris . Metabolic networks can alternatively be studied in silico with the help of genome‐scale metabolic models (GEMs), which contain all known metabolic reactions within an organism.…”