Metabolic flux analysis of Pichia pastoris carbon metabolism
combining MS- and NMR-derived 13C-labeling data
Sandra Zinke
FH-HauptbetreuerIn:
ao.Univ.Prof.DI Dr. Diethard Mattanovich
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Introduction
Pichia pastoris is a biotechnologically important microorganism. Strain improvement, rational design and optimization of cultivation conditions are of great interest to make production of recombinant proteins more efficient. Still,
the majority of flux data available for yeast origins from Saccharomyces cerevisiae. Previous studies of carbon
fluxes in P. pastoris have been done in this research group (Dep. of Chemical Engineering, Universitat Autónoma
de Barcelona) in collaboration with the NMR-lab at VTT Biotechnology in Finland. In these studies, P. pastoris was
grown in chemostat cultures on different carbon sources (glucose, glycerol and different methanol/glycerol mixtures), under different dilution rates (0,05 and 0.16 h-1). The results of this analysis were used as starting point in
this work. Previous studies of this group have allowed to establish a method of flux calculation from NMR-data [1].
Nevertheless, this method has certain limitations. NMR-data can give positional labeling information. As the methodology is based on the principle of tracing 13C-13C connectivities through a metabolic network, this positional information is significantly reduced when glycerol, a C3 source, is used as carbon source instead of glucose, a C6
source, since consequently, less information on metabolic flux mapping can be obtained. For instance, the activity
in the pentose phosphate pathway (PPP) can only be estimated from the labeling patterns of histidine hence flux
ratios through the PPP could not be determined. In addition, due to the enormous costs of 13C all previous labeling
experiments were done during one residence time and metabolic flux ratio calculations adapted with correction
factors.
In this work metabolic fluxes through pathways in the central metabolism and biosynthesis pathways of Pichia pastoris were studied in this work using a combination of two different methodologies based on 13C-labeling. In particular P. pastoris was cultivated in chemostat cultures at a dilution rate of D= 0.05h -1 on glycerol as sole carbon
source under aerobic conditions. In this experiment, NMR and MS were used as analytical tools and their results
combined.
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Results
The NMR-based methodology allowed to assess labeling patterns of the proteinogenic amino acids, whereas the
LC-MS based methodology allowed to measure the labeling patterns of several metabolites of the central carbon
metabolites, including free amino acid pools. Labeling was done over four residence times and results of all residence times compared. It could be proven that correction factors, which were used in previous studies for NMRderived data, were appropriate. Labeling states of central metabolites showed some degree of fluctuation over time
under metabolic steady state conditions, while the labeled fractions of amino acids increased and stabilized after
two or three residence times of labeling. Also, pool-sizes of cometabolites (NAD(P)H/NAD(P)) showed certain variations and could either indicate that a metabolic steady-state cannot be reached in the central metabolism or the
sampling, quenching and metabolite extraction were not adequate. It could be shown that the metabolic flux results of both NMR- and MS-derived data are comparable. The major carbon flux was measured in the glycolysis
toward the tricarboxylic acids cycle (TCA-cycle) and the TCA-cycle itself. The activity in the pentose phosphate
pathway (PPP), for which the 13C-NMR methodology could not determine the metabolic fluxes, could be measured
with the MS-based data and were, as predicted, very low. Fluxes calculated from MS-data were the same in all four
residence time though the labeling states of the metabolites were different. For the analysis of MS-data 13C-Flux
software was used. This software tool (for academic use open source), developed by the Department of Simulation
at the University of Siegen, Germany, was established as an effective analytic tool in the research group. Although
it allowed to assess fluxes in PPP, it also showed its limitations. Thus, overall, the combination of MS- & NMR-based
data allowed for more comprehensive analysis of metabolic fluxes.
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Discussion
Before starting future experiments the method of extraction used for the MS-measurement and CO2-collection require further improvement. Additionally C-Flux input data need to be adapted in order to investigate fluxes of fermentations done on different carbon sources (glucose, glycerol/methanol mixtures) and fluxes in fermentative
pathways under oxygen-limiting conditions.
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Literature
[1] Solà A., Jouhten P., Maaheimo H., Sánchez-Ferrando F., Szyperski T., Ferrer P. Metabolic flux profiling of Pichia
pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates. Microbiology.
2007 Jan;153(Pt 1):281-90
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