Angela ten Pierick

Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics.

Accurate determination of intracellular metabolite levels requires well-validated procedures for sampling and sample treatment. Several methods exist for metabolite extraction, but the literature is contradictory regarding the adequacy and performance of each technique. Using a strictly quantitative approach, we have re-evaluated five methods (hot water, HW; boiling ethanol, BE; chloroform-methanol, CM; freezing-thawing in methanol, FTM; acidic acetonitrile-methanol, AANM) for the extraction of 44 intracellular metabolites (phosphorylated intermediates, amino acids, organic acids, nucleotides) from S. cerevisiae cells. Two culture modes were investigated (batch and chemostat) to check for growth condition dependency, and three targeted platforms were employed (two LC-MS and one GC/MS) to exclude analytical bias. Additionally, for the determination of metabolite recoveries, we applied a novel approach based on addition of (13)C-labeled internal standards at different stages of sample processing. We found that the choice of extraction method can drastically affect measured metabolite levels, to an extent that for some metabolites even the direction of changes between growth conditions can be inverted. The best performances, in terms of efficacy and metabolite recoveries, were achieved with BE and CM, which yielded nearly identical levels for the metabolites analyzed. According to our results, AANM performs poorly in yeast and FTM cannot be considered adequate as an extraction method, as it does not ensure inactivation of enzymatic activity.

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

Quantitative metabolomics of microbial cultures requires well-designed sampling and quenching procedures. We successfully developed and applied a differential method to obtain a reliable set of metabolome data for Escherichia coli K12 MG1655 grown in steady-state, aerobic, glucose-limited chemostat cultures. From a rigorous analysis of the commonly applied quenching procedure based on cold aqueous methanol, it was concluded that it was not applicable because of release of a major part of the metabolites from the cells. No positive effect of buffering or increasing the ionic strength of the quenching solution was observed. Application of a differential method in principle requires metabolite measurements in total broth and filtrate for each measurement. Different methods for sampling of culture filtrate were examined, and it was found that direct filtration without cooling of the sample was the most appropriate. Analysis of culture filtrates revealed that most of the central metabolites and amino acids were present in significant amounts outside the cells. Because the turnover time of the pools of extracellular metabolites is much larger than that of the intracellular pools, the differential method should also be applicable to short-term pulse response experiments without requiring measurement of metabolites in the supernatant during the dynamic period.

A comprehensive method for the quantification of the non-oxidative pentose phosphate pathway intermediates in Saccharomyces cerevisiae by GC–IDMS

A gas chromatography isotope dilution mass spectrometry (GC-IDMS) method was developed for the quantification of the metabolites of the non-oxidative part of pentose phosphate pathway (PPP). A mid-polar GC column (Zebron ZB-AAA, 10m, film composition 50% phenyl 50% dimethyl polysiloxane) was used for the chromatographic separation of the intermediates. The optimized GC-MS procedure resulted in improved separation performances and higher sensitivities compared to previous methods. Furthermore, the use of (13)C-labeled cell extracts as internal standards improved the data quality and eliminated the need to perform a recovery check for each metabolite. The applicability of the new method was demonstrated by analyzing intracellular metabolite levels in samples derived from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae at steady state as well as following a short-term glucose pulse. The major achievements of the proposed quantitative method are the independent quantification of the epimers ribulose-5-phosphate and xylulose-5-posphate and the measurement of compounds present at very low concentrations in biological samples such as erythrose-4-phosphate and glyceraldehyde-3-phosphate.

Scale-down of penicillin production in Penicillium chrysogenum

In large-scale production reactors the combination of high broth viscosity and large broth volume leads to insufficient liquid-phase mixing, resulting in gradients in, for example, the concentrations of substrate and oxygen. This often leads to differences in productivity of the full-scale process compared with laboratory scale. In this scale-down study of penicillin production, the influence of substrate gradients on process performance and cell physiology was investigated by imposing an intermittent feeding regime on a laboratory-scale culture of a high yielding strain of Penicillium chrysogenum. It was found that penicillin production was reduced by a factor of two in the intermittently fed cultures relative to constant feed cultivations fed with the same amount of glucose per hour, while the biomass yield was the same. Measurement of the levels of the intermediates of the penicillin biosynthesis pathway, along with the enzyme levels, suggested that the reduction of the flux through the penicillin pathway is mainly the result of a lower influx into the pathway, possibly due to inhibitory levels of adenosine monophosphate and pyrophosphate and lower activating levels of adenosine triphosphate during the zero-substrate phase of each cycle of intermittent feeding.

Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary 13C flux analysis

BackgroundSeveral 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.ResultsInstationary 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).ConclusionsIn 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.

Development of quantitative metabolomics for Pichia pastoris

Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at −27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism.

Quantitative metabolomics analysis of amino acid metabolism in recombinant Pichia pastoris under different oxygen availability conditions

BackgroundEnvironmental and intrinsic stress factors can result in the global alteration of yeast physiology, as evidenced by several transcriptional studies. Hypoxia has been shown to have a beneficial effect on the expression of recombinant proteins in Pichia pastoris growing on glucose. Furthermore, transcriptional profiling analyses revealed that oxygen availability was strongly affecting ergosterol biosynthesis, central carbon metabolism and stress responses, in particular the unfolded protein response. To contribute to the better understanding of the effect and interplay of oxygen availability and foreign protein secretion on central metabolism, a first quantitative metabolomic analysis of free amino acids pools in a recombinant P. pastoris strain growing under different oxygen availability conditions has been performed.ResultsThe values obtained indicate significant variations in the intracellular amino acid pools due to different oxygen availability conditions, showing an overall increase of their size under oxygen limitation. Notably, even while foreign protein productivities were relatively low (about 40–80 μg Fab/gDCW·h), recombinant protein production was found to have a limited but significant impact on the intracellular amino acid pools, which were generally decreased in the producing strain compared with the reference strain. However, observed changes in individual amino acids pools were not correlated with their corresponding relative abundance in the recombinant protein sequence, but to the overall cell protein amino acid compositional variations.ConclusionsOverall, the results obtained, combined with previous transcriptomic and proteomic analyses provide a systematic metabolic fingerprint of the oxygen availability impact on recombinant protein production in P. pastoris.

Determination of the Cytosolic NADPH/NADP Ratio in Saccharomyces cerevisiae using Shikimate Dehydrogenase as Sensor Reaction

Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: . The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, D = 0.1 h−1) conditions, to be 22.0 ± 2.6 and 15.6 ± 0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05 ± 0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

Natural isotope correction of MS/MS measurements for metabolomics and 13C fluxomics

Fluxomics and metabolomics are crucial tools for metabolic engineering and biomedical analysis to determine the in vivo cellular state. Especially, the application of 13C isotopes allows comprehensive insights into the functional operation of cellular metabolism. Compared to single MS, tandem mass spectrometry (MS/MS) provides more detailed and accurate measurements of the metabolite enrichment patterns (tandem mass isotopomers), increasing the accuracy of metabolite concentration measurements and metabolic flux estimation. MS‐type data from isotope labeling experiments is biased by naturally occurring stable isotopes (C, H, N, O, etc.). In particular, GC–MS(/MS) requires derivatization for the usually non‐volatile intracellular metabolites introducing additional natural isotopes leading to measurements that do not directly represent the carbon labeling distribution. To make full useof LC‐ and GC–MS/MS mass isotopomer measurements, the influence of natural isotopes has to be eliminated (corrected). Our correction approach is analyzed for the two most common applications; 13C fluxomics and isotope dilution mass spectrometry (IDMS) based metabolomics. Natural isotopes can have an impact on the calculated flux distribution which strongly depends on the substrate labeling and the actual flux distribution. Second, we show that in IDMS based metabolomics natural isotopes lead to underestimated concentrations that can and should be corrected with a nonlinear calibration. Our simulations indicate that the correction for natural abundance in isotope based fluxomics and quantitative metabolomics is essential for correct data interpretation. Biotechnol. Bioeng. 2016;113: 1137–1147.

Substrate cycles in Penicillium chrysogenum quantified by isotopic non-stationary flux analysis

BackgroundPenicillium chrysogenum, the main production strain for penicillin-G, has a high content of intracellular carbohydrates, especially reduced sugars such as mannitol, arabitol, erythritol, as well as trehalose and glycogen. In previous steady state 13C wash-in experiments a delay of labeling enrichments in glycolytic intermediates was observed, which suggests turnover of storage carbohydrates. The turnover of storage pools consumes ATP which is expected to reduce the product yield for energy demanding production pathways like penicillin-G.ResultsIn this study, a 13C labeling wash-in experiment of 1 hour was performed to systematically quantify the intracellular flux distribution including eight substrate cycles. The experiments were performed using a mixed carbon source of 85% CmolGlc/CmolGlc+EtOH labeled glucose (mixture of 90% [1-13C1] and 10% [U-13C6]) and 15% ethanol [U-13C2]. It was found, that (1) also several extracellular pools are enriched with 13C labeling rapidly (trehalose, mannitol, and others), (2) the intra- to extracellular metabolite concentration ratios were comparable for a large set of metabolites while for some carbohydrates (mannitol, trehalose, and glucose) the measured ratios were much higher.ConclusionsThe fast enrichment of several extracellular carbohydrates and a concentration ratio higher than the ratio expected from cell lysis (2%) indicate active (e.g. ATP consuming) transport cycles over the cellular membrane. The flux estimation indicates, that substrate cycles account for about 52% of the gap in the ATP balance based on metabolic flux analysis.