Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production.

The production of recombinant proteins is frequently enhanced at the levels of transcription, codon usage, protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the producing cells. By incorporation of the production of a heterologous protein into a genome scale metabolic model of the yeast Pichia pastoris, the effects of overproduction were simulated and gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human superoxide dismutase (hSOD). Expression of bacterial ß-glucuronidase could be enhanced as well by most of the same genetic modifications. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, most of which changed in the same direction as predicted by the model. In vivo fluxes changed in the same direction as predicted to improve hSOD production. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy.

Accurate quantification of the redox-sensitive GSH/GSSG ratios in the yeast Pichia pastoris by HILIC-MS/MS.

A novel method for the simultaneous quantification of both glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) by hydrophilic interaction chromatography-MS/MS has been developed and is critically discussed. Internal standardization based on isotopically labeled standards for both analytes is an absolute prerequisite for accurate quantification of this redox pair. Hence, a highly efficient and selective miniaturized procedure for the synthesis of isotopically labeled GSSG from commercially available glutathione-(glycine-(13)C(2),(15)N) was established using H(2)O(2) as oxidant and NaI as catalyst. Moreover, a tool is presented to monitor and hence uncover artifactual GSSG formation due to oxidation of GSH during sample preparation, which is the main source of systematic error in GSSG analysis. For this purpose, we propose to monitor the oxidation product formed by reaction of naturally occurring GSH with the isotopically labeled GSH used as internal standard. For the determination of GSH/GSSG ratios in yeast, different extraction methods based on (1) hot extraction with aqueous, acidic, or organic solvents, (2) mechanical cell lysis, and (3) extraction at subambient temperature were investigated in terms of recovery, extraction efficiency, and artifactual formation of GSSG. Total combined uncertainties of as low as 25-30 % (coverage factor=2) for the determination of GSH/GSSG ratios without derivatization were made possible by the addition of the internal standards early in the analytical procedure (before extraction) and immediate analysis of the analytes.

Interlaboratory comparison for quantitative primary metabolite profiling in Pichia pastoris.

For the first time, an interlaboratory comparison was performed in the field of quantitative metabolite profiling in Pichia pastoris. The study was designed for the evaluation of different measurement platforms integrating different quantification strategies using internal standardization. Nineteen primary metabolites including amino acids and organic acids were selected for the study. Homogenous samples were obtained from chemostat fermentations after rapid sampling, quenching and filtration, and hot ethanol extraction. Laboratory 1 (BOKU) employed an in vivo-synthesized fully labeled U(13)C cell extracts of P. pastoris for immediate internal standardization upon cell extraction. Quantification was carried out using orthogonal reversed-phase (RP-LC) and hydrophilic interaction chromatography (HILIC) in combination with tandem mass spectrometry. Laboratory 2 (Biocrates) applied a metabolomics kit allowing fully automated, rapid derivatization, solid phase extraction and internal standardization in 96-well plates with immobilized isotopically enriched internal standards in combination with HILIC-MS-MS and RP-LC-MS-MS for organic acids and derivatized amino acids, respectively. In this study, the obtained intracellular concentrations ranged from 0.2 to 108 µmol g(-1) cell dry weight. The total combined uncertainty was estimated including uncertainty contributions from the corresponding MS-based measurement and sample preparation for each metabolite. Evidently, the uncertainty contribution of sample preparation was lower for the values obtained by laboratory 1, implementing isotope dilution upon extraction. Total combined uncertainties (K = 2) ranging from 21 to 48% and from 30 to 57% were assessed for the quantitative results obtained in laboratories 1 and 2, respectively. The major contribution arose from sample preparation, hence from repeatability precision of the extraction procedure. Finally, the laboratory intercomparison was successful as most of the investigated metabolites showed concentration levels agreeing within their total combined uncertainty, implying that accurate quantification was given. The application of isotope dilution upon extraction was an absolute prerequisite for the quantification of the redox-sensitive amino acid methionine, where no agreement between the two laboratories could be achieved.

U13C cell extract of Pichia pastoris--a powerful tool for evaluation of sample preparation in metabolomics.

Quantitative metabolic profiling is preceded by dedicated sample preparation protocols. These multistep procedures require detailed optimization and thorough validation. In this work, a uniformly (13)C-labeled (U(13)C) cell extract was used as a tool to evaluate the recoveries and repeatability precisions of the cell extraction and the extract treatment. A homogenous set of biological replicates (n = 15 samples of Pichia pastoris) was prepared for these fundamental experiments. A range of less than 30 intracellular metabolites, comprising amino acids, nucleotides, and organic acids were measured both in monoisotopic (12)C and U(13)C form by LC-MS/MS employing triple quadrupole MS, reversed phase chromatography, and HILIC. Recoveries of the sample preparation procedure ranging from 60 to 100% and repeatability precisions below 10% were obtained for most of the investigated metabolites using internal standardization approaches. Uncertainty budget calculations revealed that for this complex quantification task, in the optimum case, total combined uncertainty of 12% could be achieved. The optimum case would be represented by metabolites, easy to extract from yeast with high and precise recovery. In other cases the total combined uncertainty was significantly higher.

Observation of sorptive losses of volatile sulfur compounds during natural gas sampling.

The reason for the compound-dependent over-estimation of the recoveries of several volatile organic sulfur compounds when using a Silcosteel cylinder for sample storage as reported earlier was examined. From the different possible sources of errors that were taken into consideration, the silicone tubing, which was used to fill a standard Tedlar sample bag for calibration, was identified as the cause of the artefact. The comparison of different tubing materials showed that PTFE is the best choice since it causes only minor losses (<10%) of propyl- and butylmercaptans.

Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion.

Oxidative folding of secretory proteins in the endoplasmic reticulum (ER) is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS) caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG) in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant) proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

Oxidative protein folding and unfolded protein response elicit differing redox regulation in endoplasmic reticulum and cytosol of yeast.

Oxidative protein folding can exceed the cellular secretion machinery, inducing the unfolded protein response (UPR). Sustained endoplasmic reticulum (ER) stress leads to cell stress and disease, as described for Alzheimer, Parkinson, and diabetes mellitus, among others. It is currently assumed that the redox state of the ER is optimally balanced for formation of disulfide bonds using glutathione as the main redox buffer and that UPR causes a reduction of this organelle. The direct effect of oxidative protein folding in the ER, however, has not yet been dissected from UPR regulation. To measure in vivo redox conditions in the ER and cytosol of the yeast model organism Pichia pastoris we targeted redox-sensitive roGFP variants to the respective organelles. Thereby, we clearly demonstrate that induction of the UPR causes reduction of the cytosol in addition to ER reduction. Similarly, a more reduced redox state of the cytosol, but not of the ER, is observed during oxidative protein folding in the ER without UPR induction, as demonstrated by overexpressing genes of disulfide bond-rich secretory proteins such as porcine trypsinogen or protein disulfide isomerase (PDI1) and ER oxidase (ERO1). Cytosolic reduction seems not to be caused by the action of glutathione reductase (GLR1) and could not be compensated for by overexpression of cytosolic glutathione peroxidase (GPX1). Overexpression of GPX1 and PDI1 oxidizes the ER and increases the secretion of correctly folded proteins, demonstrating that oxidative protein folding per se is enhanced by a more oxidized ER and is counterbalanced by a more reduced cytosol. As the total glutathione concentration of these strains does not change significantly, but the ratio of GSH to GSSG is altered, either transport or redox signaling between the glutathione pools of ER and cytosol is assumed. These data clearly demonstrate that protein folding and ER stress have a severe impact on the cytosolic redox balance, which may be a major factor during development of folding-related diseases.

Solid-phase microextraction-capillary gas chromatography combined with microwave-induced plasma atomic-emission spectrometry for selenite determination.

The use of solid-phase microextraction (SPME) with gas chromatography coupled to microwave-induced plasma atomic-emission detection (GC-MIP-AED) is described for selenite [Se(IV)] speciation. Aqueous standards were derivatised with sodium tetraethyl- or tetrapropylborate and extracted by SPME. Headspace extraction of the ethyl and propyl derivatives was studied. Relevant experimental conditions were optimised, including conditions for derivatisation and extraction and those of gas chromatographic analysis. The limits of detection achieved for headspace sampling of derivatised Se(IV) were in the low ng mL(-1) range for both ethylation and propylation. When the method was applied to analysis of selenite in selenised yeast reference material results were in good agreement with the indicated values.

Combination of sorption tube sampling and thermal desorption with hollow waveguide FT-IR spectroscopy for atmospheric trace gas analysis: determination of atmospheric ethene at the lower ppb level.

The determination of organic trace gases in the ambient environment at the lower ppb level is demonstrated based on a novel technique combining sorption tube sampling on Molsieve and Carbosieve S-III, thermal desorption, and detection of the trace analyte by hollow waveguide Fourier transform infrared (HWG-FT-IR) spectroscopy. While ethene concentrations of approximately 5 ppm can be directly observed using HWG-FT-IR, enrichment factors of up to 5000 were achieved by sorption tube sampling and thermal desorption. Detection limits of approximately 1 ppb are reported. Efficient enrichment by the sampling tube is achieved due to the favorable internal volume ( approximately 0.4 cm(3) at a length of 470 mm) of the hollow waveguide serving as a miniaturized gas cell. This new method was validated for ethene by thermodesorption-cryofocusing-GC-FID as the reference method. Analytical performance has been compared for standard gas mixtures and for ethene measurements in urban air. Finally, ethene data from a sampling campaign at two alpine sites in Tyrol/Austria are presented.