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

Metabolites

Joel Jordಠ Hugo Cueto Rojas¹  Marc Carnicer²  Aljoscha Wahl¹  Pau Ferrer² 

[1] Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands; E-Mail:;Department of Chemical Engineering, Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Catalonia, Spain; E-Mails:

关键词: Pichia pastoris;    instationary ¹³C-metabolic flux analysis;    recombinant protein production;    trehalose;    metabolome;    GC-MS;    LC-MS;   

DOI  :  10.3390/metabo4020281

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.