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

1
Department of Chemical Engineering, Escola d'Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Catalonia, Spain
2
Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands
3
Kluyver Centre for Genomics of Industrial Fermentation, Delft University of Technology, 2628 BC Delft, The Netherlands
*
Authors to whom correspondence should be addressed.
Present address: LISBP–INSA, 31077 Toulouse CEDEX 04, France.
Metabolites 2014, 4(2), 281-299; https://doi.org/10.3390/metabo4020281
Received: 26 February 2014 / Revised: 8 April 2014 / Accepted: 23 April 2014 / Published: 5 May 2014
(This article belongs to the Special Issue Microbial Metabolomics)
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. View Full-Text
Keywords: Pichia pastoris; instationary 13C-metabolic flux analysis; recombinant protein production; trehalose; metabolome; GC-MS; LC-MS Pichia pastoris; instationary 13C-metabolic flux analysis; recombinant protein production; trehalose; metabolome; GC-MS; LC-MS
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Jordà, J.; Rojas, H.C.; Carnicer, M.; Wahl, A.; Ferrer, P.; Albiol, J. Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris. Metabolites 2014, 4, 281-299.

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