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

Background: Environmental 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. Results: The 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. Conclusions: Overall, 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

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

In-situ regolith seismic velocity measurement at the InSight landing site on Mars

InSight's seismometer package SEIS was placed on the surface of Mars at about 1.2 m distance from the thermal properties instrument HP3 that includes a self-hammering probe. Recording the hammering noise with SEIS provided a unique opportunity to estimate the seismic wave velocities of the shallow regolith at the landing site. However, the value of studying the seismic signals of the hammering was only realised after critical hardware decisions were already taken. Furthermore, the design and nominal operation of both SEIS and HP3 are non-ideal for such high-resolution seismic measurements. Therefore, a series of adaptations had to be implemented to operate the self-hammering probe as a controlled seismic source and SEIS as a high-frequency seismic receiver including the design of a high-precision timing and an innovative high-frequency sampling workflow. By interpreting the first-arriving seismic waves as a P-wave and identifying first-arriving S-waves by polarisation analysis, we determined effective P- and S-wave velocities of vP = 114+43-20 m/s and vS = 60+11-7 m/s, respectively, from around 2,000 hammer stroke recordings. These velocities likely represent bulk estimates for the uppermost several 10's of cm of regolith. An analysis of the P-wave incidence angles provided an independent vP/vS ratio estimate of 1.84+0.89-0.35 that compares well with the traveltime based estimate of 1.92+0.52-0.28. The low seismic velocities are consistent with those observed for low-density unconsolidated sands and are in agreement with estimates obtained by other methods

Gravitational Reference Sensor Front-End Electronics Simulator for LISA

At the ETH Zurich we are developing a modular simulator that provides a realistic simulation of the Front End Electronics (FEE) for LISA Gravitational Reference Sensor (GRS). It is based on the GRS FEE-simulator already implemented for LISA Pathfinder. It considers, in particular, the non-linearity and the critical details of hardware, such as the non-linear multiplicative noise caused by voltage reference instability, test mass charging and detailed actuation and sensing algorithms. We present the simulation modules, considering the above-mentioned features. Based on the ETH GRS FEE-simulator for LISA Pathfinder we aim to develop a modular simulator that provides a realistic simulation of GRS FEE for LISA

GRS vs. OMS Calibration in LISA Pathfinder Data Analysis

On board LISA Pathfinder spacecraft the test mass displacement along the main measurement axis is sensed in two different ways: optically and electrostatically. We have monitored the relative calibration between the two measurements during the mission science phase. The trend sensitivity of the relative calibration has been computed for different physical parameters, such as temperature, magnetic field, test mass bias voltage and current

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

Background: Environmental 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. Results: The 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. Conclusions: Overall, 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

Development of an accurate method for intracellular metabolome analysis in Escherichia coli for in vivo kinetic analysis

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

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