Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris

Autor: Joel Jordà, Hugo Cueto Rojas, Marc Carnicer, Aljoscha Wahl, Pau Ferrer, Joan Albiol
Jazyk: angličtina
Rok vydání: 2014
Předmět:
Zdroj: Metabolites, Vol 4, Iss 2, Pp 281-299 (2014)
Druh dokumentu: article
ISSN: 2218-1989
DOI: 10.3390/metabo4020281
Popis: Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA) using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol) on P. pastoris central carbon metabolism. Higher oxygen uptake and CO2 production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by 13C-based metabolic flux analysis. In particular, the flux through the methanol oxidation pathway and the TCA cycle was increased in the Rol-producing strain compared to the reference strain. Next to changes in the flux distribution, significant variations in intracellular metabolite concentrations were observed. Most notably, the pools of trehalose, which is related to cellular stress response, and xylose, which is linked to methanol assimilation, were significantly increased in the recombinant strain.
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