Joan Albiol

The Effect of Temperature on the Proteome of Recombinant Pichia pastoris

The impact of environmental factors on the productivity of yeast cells is poorly investigated so far. Therefore, it is a major concern to improve the understanding of cellular physiology of microbial protein production hosts, including the methylotrophic yeast Pichia pastoris. Two-Dimensional Fluorescence Difference Gel electrophoresis and protein identification via mass spectrometry were applied to analyze the impact of cultivation temperature on the physiology of a heterologous protein secreting P. pastoris strain. Furthermore, specific productivity was monitored and fluxes through the central carbon metabolism were calculated. Chemostat culture conditions were applied to assess the adaption to different growth temperatures (20, 25, 30 degrees C) at steady-state conditions. Many important cellular processes, including the central carbon metabolism, stress response and protein folding are affected by changing the growth temperature. A 3-fold increased specific productivity at lower cultivation temperature for an antibody Fab fragment was accompanied by a reduced flux through the TCA-cycle, reduced levels of proteins involved in oxidative stress response and lower cellular levels of molecular chaperones. These data indicate that folding stress is generally decreased at lower cultivation temperatures, enabling more efficient heterologous protein secretion in P. pastoris host cells.

A multi-level study of recombinant Pichia pastoris in different oxygen conditions

BackgroundYeasts are attractive expression platforms for many recombinant proteins, and there is evidence for an important interrelation between the protein secretion machinery and environmental stresses. While adaptive responses to such stresses are extensively studied in Saccharomyces cerevisiae, little is known about their impact on the physiology of Pichia pastoris. We have recently reported a beneficial effect of hypoxia on recombinant Fab secretion in P. pastoris chemostat cultivations. As a consequence, a systems biology approach was used to comprehensively identify cellular adaptations to low oxygen availability and the additional burden of protein production. Gene expression profiling was combined with proteomic analyses and the 13C isotope labelling based experimental determination of metabolic fluxes in the central carbon metabolism.ResultsThe physiological adaptation of P. pastoris to hypoxia showed distinct traits in relation to the model yeast S. cerevisiae. There was a positive correlation between the transcriptomic, proteomic and metabolic fluxes adaptation of P. pastoris core metabolism to hypoxia, yielding clear evidence of a strong transcriptional regulation component of key pathways such as glycolysis, pentose phosphate pathway and TCA cycle. In addition, the adaptation to reduced oxygen revealed important changes in lipid metabolism, stress responses, as well as protein folding and trafficking.ConclusionsThis systems level study helped to understand the physiological adaptations of cellular mechanisms to low oxygen availability in a recombinant P. pastoris strain. Remarkably, the integration of data from three different levels allowed for the identification of differences in the regulation of the core metabolism between P. pastoris and S. cerevisiae. Detailed comparative analysis of the transcriptomic data also led to new insights into the gene expression profiles of several cellular processes that are not only susceptible to low oxygen concentrations, but might also contribute to enhanced protein secretion.

Macromolecular and elemental composition analysis and extracellular metabolite balances of Pichia pastoris growing at different oxygen levels

BackgroundAnalysis of the cell operation at the metabolic level requires collecting data of different types and to determine their confidence level. In addition, the acquired information has to be combined in order to obtain a consistent operational view. In the case of Pichia pastoris, information of its biomass composition at macromolecular and elemental level is scarce particularly when different environmental conditions, such as oxygen availability or, genetic backgrounds (e.g. recombinant protein production vs. non production conditions) are compared.ResultsP. pastoris cells growing in carbon-limited chemostat cultures under different oxygenation conditions (% O2 in the bioreactor inlet gas: 21%, 11% and 8%, corresponding to normoxic, oxygen-limiting and hypoxic conditions, respectively), as well as under recombinant protein (antibody fragment, Fab) producing and non-producing conditions, were analyzed from different points of view. On the one hand, the macromolecular and elemental composition of the biomass was measured using different techniques at the different experimental conditions and proper reconciliation techniques were applied for gross error detection of the measured substrates and products conversion rates. On the other hand, fermentation data was analyzed applying elemental mass balances. This allowed detecting a previously missed by-product secreted under hypoxic conditions, identified as arabinitol (aka. arabitol). After identification of this C5 sugar alcohol as a fermentation by-product, the mass balances of the fermentation experiments were validated.ConclusionsAfter application of a range of analytical and statistical techniques, a consistent view of growth parameters and compositional data of P. pastoris cells growing under different oxygenation conditions was obtained. The obtained data provides a first view of the effects of oxygen limitation on the physiology of this microorganism, while recombinant Fab production seems to have little or no impact at this level of analysis. Furthermore, the results will be highly useful in other complementary quantitative studies of P. pastoris physiology, such as metabolic flux analysis.

Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

BackgroundThe methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production.ResultsThe metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source.The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates.ConclusionsOverall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed methanol:multicarbon sources has been implemented, thus providing a new tool for the investigation of the relationships between central metabolism and protein production. Specifically, the study points at a limited but significant impact of the conformational stress associated to secretion of recombinant proteins on the central metabolism, occurring even at modest production levels.

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.

Modeling photoheterotrophic growth kinetics of Rhodospirillum rubrum in rectangular photobioreactors

Based on a previously established model for radiant light transfer in photobioreactors (PBR), taking into account absorption and scattering of light, a new knowledge model for coupling radiant light energy available and local growth kinetics in PBRs for the photoheterotrophic bacteria Rhodospirillum rubrum is discussed. A revised method is presented for the calculation of the absorption and scattering coefficients. The specific characteristics of the electron‐transfer chains in such microorganisms leads to definition of three different metabolic zones in the PBR, explaining the behavior of mean kinetics observed in a wide range of incident light fluxes. The model is validated in rectangular PBRs for five different carbon sources and proved robust and fully predictive. This approach can be considered for simulation and model‐based predictive control of PBRs cultivating photoheterotrophic microorganisms.

Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived 13C-labelling data from proteinogenic amino acids

The yeast Pichia pastoris has emerged as one of the most promising yeast cell factories for the production of heterologous proteins. The readily available genetic tools and the ease of high-cell density cultivations using methanol or glycerol/methanol mixtures are among the key factors for this development. Previous studies have shown that the use of mixed feeds of glycerol and methanol seem to alleviate the metabolic burden derived from protein production, allowing for higher specific and volumetric process productivities. However, initial studies of glycerol/methanol co-metabolism in P. pastoris by classical metabolic flux analyses using (13)C-derived Metabolic Flux Ratio (METAFoR) constraints were hampered by the reduced labelling information obtained when using C3:C1 substrate mixtures in relation to the conventional C6 substrate, that is, glucose. In this study, carbon flux distributions through the central metabolic pathways in glycerol/methanol co-assimilation conditions have been further characterised using biosynthetically directed fractional (13)C labelling. In particular, metabolic flux distributions were obtained under 3 different glycerol/methanol ratios and growth rates by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids using the software tool (13)CFlux2. Specifically, cells were grown aerobically in chemostat cultures fed with 80:20, 60:40 and 40:60 (w:w) glycerol/methanol mixtures at two dilutions rates (0.05 hour(-1) and 0.16 hour(-1)), allowing to obtain additional data (biomass composition and extracellular fluxes) to complement pre-existing datasets. The performed (13)C-MFA reveals a significant redistribution of carbon fluxes in the central carbon metabolism as a result of the shift in the dilution rate, while the ratio of carbon sources has a lower impact on carbon flux distribution in cells growing at the same dilution rate. At low growth rate, the percentage of methanol directly dissimilated to CO2 ranges between 50% and 70%. At high growth rate the methanol is completely dissimilated to CO2 by the direct pathway, in the two conditions of highest methanol content.

Metabolic Flux Analysis during the Exponential Growth Phase of Saccharomyces cerevisiae in Wine Fermentations

As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.

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.