Abstract
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.
Highlights
Pichia pastoris has become one of the most commonly used expression systems for recombinant protein production during recent years [1,2,3,4]
P. pastoris cells (X-33/ROL) producing the secreted protein Rhizopus oryzae lipase (Rol) were grown in aerobic, glucose/methanol limited chemostat cultures at a dilution rate of 0.09 h 1
Once the steady state was obtained, the consumption rates of glucose, methanol and oxygen and the production rates of biomass and carbon dioxide were calculated from measurements of biomass (dry cell weight (DCW)), residual glucose and the concentration of oxygen and carbon dioxide in the off-gas line (Table 1)
Summary
Pichia pastoris has become one of the most commonly used expression systems for recombinant protein production during recent years [1,2,3,4]. A close link between protein production and energy metabolism is suggested by the successful strategy to supply the cell with substrate mixtures like mixing methanol with a multi-carbon source such as sorbitol or glycerol [10]. By using such strategies, the production rate of recombinant protein is significantly increased during high cell density cultivations, [11,12,13,14]. We quantify the impact of Rol production and secretion on central carbon metabolism using metabolomics and 13C transient flux analysis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
