Christina Haberhauer-Troyer

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 β-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.

Evaluation of solid-phase microextraction for sampling of volatile organic sulfur compounds in air for subsequent gas chromatographic analysis with atomic emission detection

Abstract The use of solid-phase microextraction (SPME) followed by GC–AED (atomic emission detection) for the analysis of volatile organic sulfur compounds (methanethiol, dimethyl sulfide, isopropanethiol and isobutanethiol) in spiked air samples was investigated. Gaseous standard mixtures were generated by means of a permeation apparatus with stopped flow facilities to permit sampling of the analytes with the SPME fiber. Detection limits between 4 ppt for dimethyl sulfide and isobutanethiol and 50 ppt (v/v) for methanethiol were achieved for extraction with the Carboxen–PDMS (polydimethylsiloxane) fiber followed by GC–AED analysis. The comparison of the performance of the 100 μm PDMS and the 75 μm Carboxen–PDMS fiber coating demonstrates the superiority of the latter in terms of sensitivity and repeatability. Despite the principal applicability of SPME to sampling of organosulfur compounds, artifacts are observed during analysis. Furthermore, the low storage stability, the dependence of the extraction efficiency on the relative humidity and the pronounced differences in sensitivity between fibers limit the usefulness of the method for quantitative on-site analysis.

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.

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.

Investigation of the storage stability of selected volatile sulfur compounds in different sampling containers

The suitability of various sample containers (i.e. standard Tedlar sample bags, black/clear layered Tedlar sample bags and Silcosteel sample cylinders) was examined for a gaseous multicomponent standard containing methylmercaptan, ethylmercaptan, dimethyl sulfide, ethylmethyl sulfide, 2-propylmercaptan, 1-propylmercaptan, 2-butylmercaptan, diethyl sulfide and 1-butylmercaptan (1 mg/m3 each in nitrogen). In the black/clear layered Tedlar sample bags, significant losses (up to 10% for methylmercaptan as the most critical component) were observed after 2 days, whereas in the standard Tedlar sample bags the recovery of methylmercaptan was approximately 90% even after 1 week. The Silcosteel sample cylinders were suitable for sampling of volatile sulfur compounds with respect to the stability of the analytes, but the recoveries exceeded 100% especially for the higher boiling compounds, which was attributed to enrichment effects on parts of the sampling system.

Comparison of different chloroformates for the derivatisation of seleno amino acids for gas chromatographic analysis

Three chloroformate reagents, ethyl chloroformate (ECF), methyl chloroformate (MCF) and menthyl chloroformate (MenCF), have been used for the derivatisation of seleno amino acids and their performance was compared. Chromatographic parameters and the inertness of the different instrumental configurations used (gas chromatography-atomic emission detection (GC-AED), and GC-MS) were shown to have a significant influence on the detection of various seleno amino acids (selenomethione, selenoethione and selenocysteine) and some sulphur-containing amino acids (methionine, cysteine, cystine and methylcysteine) which were included in the experiments for comparison. Methyl chloroformate was the preferred derivatisation reagent, since it generally performed best in terms of derivatisation yield and reproducibility and also showed less significant conditioning effects than ethyl chloroformate. Methyl and ethyl chloroformate derivatives of selenomethionine, selenoethionine, cysteine and methionine were detectable, while the detection of the menthyl chloroformate derivatives of selenocystine and cystine was not reproducible. Overall efficiencies for the determination of selenomethionine and selenoethionine from aqueous extracts ranged from 40 to 100% for methyl chloroformate, over 30-75% for ethyl chloroformate to 15-70% for menthyl chloroformate for different series measured over a period of months. The relative standard deviation of the method for the methyl and menthyl chloroformate derivatisation ranged from 7 to 13% without internal standard and was improved to 2% for the determination of selenomethionine using selenoethionine as internal standard. This indicates that, despite the limited reproducibility of the method, its repeatability is good enough to allow accurate determination of seleno amino acids, which was also demonstrated by the analysis of selenium supplementation tablets for human diet that contained selenomethionine.

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.

Investigation of membrane dryers and evaluation of a new ozone scrubbing material for the sampling of organosulphur compounds in air.

The applicability of two different types of Nafion membrane dryers (based on counter-current flow and desiccant drying) and of a new ozone scrubbing material, polyphenylene sulphide wool (noXon-S), to adsorptive sampling of selected volatile sulphur compounds (methanethiol, dimethyl sulphide, isopropanethiol and isobutanethiol) is investigated at the low ppb (v/v) level (1-5 ppb). No analyte losses occur with either type of dryer at relative humidities (RH) of < or = 50%, while at higher RH values particularly the thiols tend to be lost (between 6 and 32%) even after conditioning. The actual losses depend more on the state of the individual permeation membrane rather than on the type of dryer. NoXon-S is a highly suitable ozone scrubber material for sulphur compounds since it efficiently removes ozone without retainment of the analytes and without the formation of blanks or artefacts from the scrubber material. The combined use of a Nafion membrane dryer and a noXon-S ozone scrubber is thus recommended for artifact-free sampling of sulphur compounds.