Per citar aquest document: http://ddd.uab.cat/record/125688
Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary ¹³C flux analysis
Jordà Murria, Joel (Universitat Autònoma de Barcelona. Departament d’Enginyeria Química)
Suárez, Camilo (Delft University of Technology. Department of Biotechnology)
Carnicer Heras, Marc (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
ten Pierick, Angela (Delft University of Technology. Department of Biotechnology)
Heijnen, Joseph J. (Delft University of Technology. Department of Biotechnology)
van Gulik, Walter (Delft University of Technology. Department of Biotechnology)
Ferrer i Alegre, Pau (Universitat Autònoma de Barcelona. Departament d’Enginyeria Química)
Albiol i Sala, Joan (Universitat Autonoma de Barcelona. Departament d'Enginyeria Química)
Aljoscha Wahl (Delft University of Technology. Department of Biotechnology)

Data: 2013
Resum: Background: Several studies have shown that the utilization of mixed carbon feeds instead of methanol as sole carbon source is beneficial for protein production with the methylotrophic yeast Pichia pastoris. In particular, growth under mixed feed conditions appears to alleviate the metabolic burden related to stress responses triggered by protein overproduction and secretion. Yet, detailed analysis of the metabolome and fluxome under mixed carbon source metabolizing conditions are missing. To obtain a detailed flux distribution of central carbon metabolism, including the pentose phosphate pathway under methanol-glucose conditions, we have applied metabolomics and instationary 13C flux analysis in chemostat cultivations. Results: Instationary 13C-based metabolic flux analysis using GC-MS and LC-MS measurements in time allowed for an accurate mapping of metabolic fluxes of glycolysis, pentose phosphate and methanol assimilation pathways. Compared to previous results from NMR-derived stationary state labelling data (proteinogenic amino acids, METAFoR) more fluxes could be determined with higher accuracy. Furthermore, using a thermodynamic metabolic network analysis the metabolite measurements and metabolic flux directions were validated. Notably, the concentration of several metabolites of the upper glycolysis and pentose phosphate pathway increased under glucose-methanol feeding compared to the reference glucose conditions, indicating a shift in the thermodynamic driving forces. Conversely, the extracellular concentrations of all measured metabolites were lower compared with the corresponding exometabolome of glucose-grown P. pastoris cells. The instationary 13C flux analysis resulted in fluxes comparable to previously obtained from NMR datasets of proteinogenic amino acids, but allowed several additional insights. Specifically, i) in vivo metabolic flux estimations were expanded to a larger metabolic network e. g. by including trehalose recycling, which accounted for about 1. 5% of the glucose uptake rate; ii) the reversibility of glycolytic/gluconeogenesis, TCA cycle and pentose phosphate pathways reactions was estimated, revealing a significant gluconeogenic flux from the dihydroxyacetone phosphate/glyceraldehydes phosphate pool to glucose-6P. The origin of this finding could be carbon recycling from the methanol assimilatory pathway to the pentose phosphate pool. Additionally, high exchange fluxes of oxaloacetate with aspartate as well as malate indicated amino acid pool buffering and the activity of the malate/Asp shuttle; iii) the ratio of methanol oxidation vs utilization appeared to be lower (54 vs 79% assimilated methanol directly oxidized to CO2). Conclusions: In summary, the application of instationary 13C-based metabolic flux analysis to P. pastoris provides an experimental framework with improved capabilities to explore the regulation of the carbon and energy metabolism of this yeast, particularly for the case of methanol and multicarbon source metabolism.
Drets: Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original. Creative Commons
Llengua: Anglès
Document: article ; recerca ; publishedVersion
Matèria: Pichia pastoris ; Instationary 13C-metabolic flux analysis ; GC-MS ; LC-MS
Publicat a: BMC systems biology, Vol. 7, Num. 17 (2013) , p. 1-16, ISSN 1752-0509

DOI: 10.1186/1752-0509-7-17


16 p, 760.8 KB

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