Conversion of CO2 into organic acids by engineered autotrophic yeast.

The increase of CO2 emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO2 as a feedstock gains an extensive importance to establish CO2-neutral or CO2-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO2 as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. 13C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO2 as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L-1, while balancing the expression of key genes and modifying screening conditions led to 2 g L-1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO2 uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO2, to support sustainability of industrial bioprocesses.

Protein production dynamics and physiological adaptation of recombinant Komagataella phaffii at near-zero growth rates.

BACKGROUND: Specific productivity (qP) in yeast correlates with growth, typically peaking at intermediate or maximum specific growth rates (µ). Understanding the factors limiting productivity at extremely low µ might reveal decoupling strategies, but knowledge of production dynamics and physiology in such conditions is scarce. Retentostats, a type of continuous cultivation, enable the well-controlled transition to near-zero µ through the combined retention of biomass and limited substrate supply. Recombinant Komagataella phaffii (syn Pichia pastoris) secreting a bivalent single domain antibody (VHH) was cultivated in aerobic, glucose-limited retentostats to investigate recombinant protein production dynamics and broaden our understanding of relevant physiological adaptations at near-zero growth conditions. RESULTS: By the end of the retentostat cultivation, doubling times of approx. two months were reached, corresponding to µ = 0.00047 h-1. Despite these extremely slow growth rates, the proportion of viable cells remained high, and de novo synthesis and secretion of the VHH were observed. The average qP at the end of the retentostat was estimated at 0.019 mg g-1 h-1. Transcriptomics indicated that genes involved in protein biosynthesis were only moderately downregulated towards zero growth, while secretory pathway genes were mostly regulated in a manner seemingly detrimental to protein secretion. Adaptation to near-zero growth conditions of recombinant K. phaffii resulted in significant changes in the total protein, RNA, DNA and lipid content, and lipidomics revealed a complex adaptation pattern regarding the lipid class composition. The higher abundance of storage lipids as well as storage carbohydrates indicates that the cells are preparing for long-term survival. CONCLUSIONS: In conclusion, retentostat cultivation proved to be a valuable tool to identify potential engineering targets to decouple growth and protein production and gain important insights into the physiological adaptation of K. phaffii to near-zero growth conditions.

Genotypic and phenotypic diversity among Komagataella species reveals a hidden pathway for xylose utilization.

BACKGROUND: The yeast genus Komagataella currently consists of seven methylotrophic species isolated from tree environments. Well-characterized strains of K. phaffii and K. pastoris are important hosts for biotechnological applications, but the potential of other species from the genus remains largely unexplored. In this study, we characterized 25 natural isolates from all seven described Komagataella species to identify interesting traits and provide a comprehensive overview of the genotypic and phenotypic diversity available within this genus. RESULTS: Growth tests on different carbon sources and in the presence of stressors at two different temperatures allowed us to identify strains with differences in tolerance to high pH, high temperature, and growth on xylose. As Komagataella species are generally not considered xylose-utilizing yeasts, xylose assimilation was characterized in detail. Growth assays, enzyme activity measurements and 13C labeling confirmed the ability of K. phaffii to utilize D-xylose via the oxidoreductase pathway. In addition, we performed long-read whole-genome sequencing to generate genome assemblies of all Komagataella species type strains and additional K. phaffii and K. pastoris isolates for comparative analysis. All sequenced genomes have a similar size and share 83-99% average sequence identity. Genome structure analysis showed that K. pastoris and K. ulmi share the same rearrangements in difference to K. phaffii, while the genome structure of K. kurtzmanii is similar to K. phaffii. The genomes of the other, more distant species showed a larger number of structural differences. Moreover, we used the newly assembled genomes to identify putative orthologs of important xylose-related genes in the different Komagataella species. CONCLUSIONS: By characterizing the phenotypes of 25 natural Komagataella isolates, we could identify strains with improved growth on different relevant carbon sources and stress conditions. Our data on the phenotypic and genotypic diversity will provide the basis for the use of so-far neglected Komagataella strains with interesting characteristics and the elucidation of the genetic determinants of improved growth and stress tolerance for targeted strain improvement.

Yeast-based reference materials for quantitative metabolomics.

We introduce a new concept of yeast-derived biological matrix reference material for metabolomics research relying on in vivo synthesis of a defined biomass, standardized extraction followed by absolute quantification with isotope dilution. The yeast Pichia pastoris was grown using full control- and online monitoring fed-batch fermentations followed by fast cold methanol quenching and boiling ethanol extraction. Dried extracts served for the quantification campaign. A metabolite panel of the evolutionarily conserved primary metabolome (amino acids, nucleotides, organic acids, and metabolites of the central carbon metabolism) was absolutely quantified by isotope dilution utilizing uniformly labeled 13C-yeast-based internal standards. The study involved two independent laboratories employing complementary mass spectrometry platforms, namely hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Homogeneity, stability tests (on a panel of >70 metabolites over a period of 6 months), and excellent biological repeatability of independent fermentations over a period of 2 years showed the feasibility of producing biological reference materials on demand. The obtained control ranges proved to be fit for purpose as they were either superior or comparable to the established reference materials in the field.

Beyond alcohol oxidase: the methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2.

Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut- phenotype. The Mut- phenotype is commonly regarded as unable to utilize methanol. In contrast to the literature, we found that the Mut- strain can consume methanol. This ability was based on the promiscuous activity of alcohol dehydrogenase Adh2, an enzyme ubiquitously found in yeast and normally responsible for ethanol consumption and production. Using 13C labeled methanol as substrate we could show that to the largest part methanol is dissimilated to CO2 and a small part is incorporated into metabolites, the biomass, and the secreted recombinant protein. Overexpression of the ADH2 gene in K. phaffii Mut- increased both the specific methanol uptake rate and recombinant protein production, even though the strain was still unable to grow. These findings imply that thermodynamic and kinetic constraints of the dehydrogenase reaction facilitated the evolution towards alcohol oxidase-based methanol metabolism in yeast.

Selective and Accurate Quantification of N-Acetylglucosamine in Biotechnological Cell Samples via GC-MS/MS and GC-TOFMS.

N-Acetylglucosamine is a key component of bacterial and fungal cell walls and of the extracellular matrix of animal cells. It plays a variety of roles at the cell surface structure and is under discussion to be involved in signaling pathways. The presence of a number of N-acetylhexosamine stereoisomers in samples of biological or biotechnological origin demands for dedicated high efficiency separation methods, due to identical exact mass and similar fragmentation patterns of the stereoisomers. Gas chromatography offers high sample capacity, separation efficiency, and precision under repeatability conditions of measurement, which is a necessity for the analysis of low abundant stereoisomers in biological samples. Automated online derivatization facilitates to overcome the main obstacle for the use of gas chromatography in metabolomics, namely, the derivatization of polar metabolites prior to analysis. Using alkoximation and subsequent trimethylsilylation, carbohydrates and their derivatives are known to show several derivatives, since derivatization is incomplete as well as highly matrix dependent inherent to the high number of functional groups present in carbohydrates. A method based on efficient separation of ethoximated and trimethylsilylated N-acetylglucosamines was developed. Accurate absolute quantification is enabled using biologically derived 13C labeled internal standards eliminating systematic errors related to sample pretreatment and analysis. Due to the lack of certified reference materials, a methodological comparison between tandem and time-of-flight mass spectrometric instrumentation was performed for mass spectrometric assessment of trueness. Both methods showed limits of detection in the lower femtomol range. The methods were applied to biological samples of Penicillium chrysogenum cultivations with different matrices revealing excellent agreement of both mass spectrometric techniques.

What CHO is made of: Variations in the biomass composition of Chinese hamster ovary cell lines.

BACKGROUND: Cell line-specific, genome-scale metabolic models enable rigorous and systematic in silico investigation of cellular metabolism. Such models have recently become available for Chinese hamster ovary (CHO) cells. However, a key ingredient, namely an experimentally validated biomass function that summarizes the cellular composition, was so far missing. Here, we close this gap by providing extensive experimental data on the biomass composition of 13 parental and producer CHO cell lines under various conditions. RESULTS: We report total protein, lipid, DNA, RNA and carbohydrate content, cell dry mass, and detailed protein and lipid composition. Furthermore, we present meticulous data on exchange rates between cells and environment and provide detailed experimental protocols on how to determine all of the above. The biomass composition is converted into cell line- and condition-specific biomass functions for use in cell line-specific, genome-scale metabolic models of CHO. Finally, flux balance analysis (FBA) is used to demonstrate consistency between in silico predictions and experimental analysis. CONCLUSIONS: Our study reveals a strong variability of the total protein content and cell dry mass across cell lines. However, the relative amino acid composition is independent of the cell line and condition and thus needs not be explicitly measured for each new cell line. In contrast, the lipid composition is strongly influenced by the growth media and thus will have to be determined in each case. These cell line-specific variations in biomass composition have a small impact on growth rate predictions with FBA, as inaccuracies in the predictions are rather dominated by inaccuracies in the exchange rate spectra. Cell-specific biomass variations only become important if the experimental errors in the exchange rate spectra drop below twenty percent.

Systems-level organization of yeast methylotrophic lifestyle.

BACKGROUND: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. RESULTS: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. CONCLUSIONS: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology.

Metabolomics sampling of Pichia pastoris revisited: rapid filtration prevents metabolite loss during quenching.

Metabolomics can be defined as the quantitative assessment of a large number of metabolites of a biological system. A prerequisite for the accurate determination of intracellular metabolite concentrations is a reliable and reproducible sample preparation method, which needs to be optimized for each organism individually. Here, we compare the performance of rapid filtration and centrifugation after quenching of Pichia pastoris cells in cold methanol. During incubation in the quenching solution, metabolites are lost from the cells with a half-life of 70-180 min. Metabolites with lower molecular weights showed lower half-lifes compared to metabolites with higher molecular weight. Rapid filtration within 2 min after quenching leads to only minor losses below 2%, and is thus the preferred method for cell separation.

Isotopologue analysis of sugar phosphates in yeast cell extracts by gas chromatography chemical ionization time-of-flight mass spectrometry.

Metabolic flux analysis is based on the measurement of isotopologue ratios. In this work, a new GC-MS-based method was introduced enabling accurate determination of isotopologue distributions of sugar phosphates in cell extracts. A GC-TOFMS procedure was developed involving a two-step online derivatization (ethoximation followed by trimethylsilylation) offering high mass resolution, high mass accuracy and the potential of retrospective data analysis typical for TOFMS. The information loss due to fragmentation intrinsic for isotopologue analysis by electron ionization could be overcome by chemical ionization with methane. A thorough optimization regarding pressure of the reaction gas, emission current, electron energy and temperature of the ion source was carried out. For a substantial panel of sugar phosphates both of the glycolysis and the pentose phosphate pathway, sensitive determination of the protonated intact molecular ions together with low abundance fragment ions was successfully achieved. The developed method was evaluated for analysis of Pichia pastoris cell extracts. The measured isotopologue ratios were in the range of 55:1-2:1. The comparison of the experimental isotopologue fractions with the theoretical fractions was excellent, revealing a maximum bias of 4.6% and an average bias of 1.4%.

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.

A native phosphoglycolate salvage pathway of the synthetic autotrophic yeast Komagataella phaffii.

Synthetic autotrophs can serve as chassis strains for bioproduction from CO2 as a feedstock to take measures against the climate crisis. Integration of the Calvin-Benson-Bassham (CBB) cycle into the methylotrophic yeast Komagataella phaffii (Pichia pastoris) enabled it to use CO2 as the sole carbon source. The key enzyme in this cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzing the carboxylation step. However, this enzyme is error prone to perform an oxygenation reaction leading to the production of toxic 2-phosphoglycolate. Native autotrophs have evolved different recycling pathways for 2-phosphoglycolate. However, for synthetic autotrophs, no information is available for the existence of such pathways. Deletion of CYB2 in the autotrophic K. phaffii strain led to the accumulation of glycolate, an intermediate in phosphoglycolate salvage pathways, suggesting that such a pathway is enabled by native K. phaffii enzymes. 13C tracer analysis with labeled glycolate indicated that the yeast pathway recycling phosphoglycolate is similar to the plant salvage pathway. This orthogonal yeast pathway may serve as a sensor for RuBisCO oxygenation, and as an engineering target to boost autotrophic growth rates in K. phaffii.

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.

Spoilage markers for freshwater fish: A comprehensive workflow for non-targeted analysis of VOCs using DHS-GC-HRMS.

Changes of volatile organic compounds (VOCs) patterns during 6 days of storage at +4 °C were investigated in different freshwater fish species, namely carp and trout, using dynamic headspace gas chromatography time-of-flight mass spectrometry (DHS-GC-TOFMS). DHS parameters were systematically optimized to establish optimum extraction and pre-concentration of VOCs. Moreover, different sample preparation methods were tested: mincing with a manual meat grinder, as well as mincing plus homogenization with a handheld homogenizer both without and with water addition. The addition of water during sample preparation led to pronounced changes of the volatile profiles, depending on the molecular structure and lipophilicity of the analytes, resulting in losses of up to 98 % of more lipophilic compounds (logP > 3). The optimized method was applied to trout and carp. Trout samples of different storage days were compared using univariate (Mann-Whitney U test, fold change calculation) and multivariate (OPLS-DA) statistics. 37 potential spoilage markers were selected; for 11 compounds identity could be confirmed via measurement of authentic standards and 10 compounds were identified by library spectrum match. 22 compounds were also found to be statistically significant spoilage markers in carp. Merging results of the different statistical approaches, the list of 37 compounds could be narrowed down to the 14 most suitable for trout spoilage assessment. This study comprises a systematic evaluation of the capabilities of DHS-GC coupled to high-resolution (HR) MS for studying spoilage in different freshwater fish species, including a comprehensive data evaluation workflow.

The oxygen-tolerant reductive glycine pathway assimilates methanol, formate and CO2 in the yeast Komagataella phaffii.

The current climatic change is predominantly driven by excessive anthropogenic CO2 emissions. As industrial bioprocesses primarily depend on food-competing organic feedstocks or fossil raw materials, CO2 co-assimilation or the use of CO2-derived methanol or formate as carbon sources are considered pathbreaking contributions to solving this global problem. The number of industrially-relevant microorganisms that can use these two carbon sources is limited, and even fewer can concurrently co-assimilate CO2. Here, we search for alternative native methanol and formate assimilation pathways that co-assimilate CO2 in the industrially-relevant methylotrophic yeast Komagataella phaffii (Pichia pastoris). Using 13C-tracer-based metabolomic techniques and metabolic engineering approaches, we discover and confirm a growth supporting pathway based on native enzymes that can perform all three assimilations: namely, the oxygen-tolerant reductive glycine pathway. This finding paves the way towards metabolic engineering of formate and CO2 utilisation to produce proteins, biomass, or chemicals in yeast.

Importance of hydraulic travel time for the evaluation of organic compounds removal in bank filtration.

The growing global demand for drinking water is driving both the diversification of water supply sources and their sustainability. River bank filtration (RBF) is an excellent option since it strongly reduces the extent of treatment steps compared to direct usage of surface water. Organic micropollutants (e.g. pharmaceuticals) are widely recognized as a hazard in drinking water production from surface water. Due to their potentially high mobility, stability, bioaccumulation and persistency, these substances can pass through RBF-systems. Scientific studies on compound removal and attenuation efficiency of RBF rely on the knowledge of travel time to compare concentrations in the river to the ones in the bank filtrate since water quality in rivers can change rapidly. However, bank filtrate samples represent a mixture of water with different travel times as the flow paths vary. This has not yet been considered in studies of bank filtration removal efficiency for organic micro pollutants. Here we present a method that considers the residence-time distribution of the bank filtrate sample obtained by groundwater modelling to evaluate the removal efficiency of RBF for organic micropollutants. The method was tested in a comprehensive study with 50 samples taken over a one-year-period at a river bank filtration site in Vienna (Austria). Our findings revealed that better coverage of varying river water quality (higher sampling frequency during the period of infiltration) resulted not only in a higher number of compounds considered as removed but also significantly reduced the number of compounds considered to have formed during the RBF process. The application of the presented method indicated that RBF is very effective in removing organic micropollutants. Considering different travel times will provide better models and a better understanding of the potential of RBF for pollutant removal and thus supports its safe application as a solution to the growing demand for drinking water.

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.

Non-targeted analysis with high-resolution mass spectrometry for investigation of riverbank filtration processes.

A fully non-targeted analytical workflow for the investigation of a riverbank filtration site located at the river Danube has been developed and applied. Variations of compound intensities at different sampling locations of the riverbank filtration site and, for a single production well, over a monitoring period of one year have been investigated using liquid chromatography combined with time-of-flight-mass spectrometry followed by evaluation via non-targeted data analysis. Internal standardization and appropriate quality control strategies have been implemented into the workflow for reduction of possible methodological biases influencing data interpretation. Emphasis was placed on the assessment of different blank elimination steps and the final blank elimination strategy is reported. The spatial study of the selected riverbank filtration site revealed a homogenous composition of the filtered water sampled at 11 different locations across the 32,000 m2 site, except for one sampling location in a zone of the aquifer, which was only weakly connected to the well field in terms of hydrogeological conditions. The examination of time-dependent changes of the composition of surface and groundwater obtained at the riverbank filtration system revealed that the non-targeted workflow is fit-for-purpose regarding the assessment the stability of filtration efficiency and compound residence time in the riverbank filtration compartment. In total, 677 compounds were selected for the investigation of the time-dependent variations of the filtration process. Analysis of the signal intensities of these compounds revealed that the riverbank filtration is significantly reducing the intensity and number of compounds present in surface water over a wide polarity range. In addition, the method enabled the determination of compound residence times in the riverbank filtration system ranging from 5 to 7 days.

The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2.

The methylotrophic yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals. Like most biotechnological production hosts, P. pastoris is heterotrophic and grows on organic feedstocks that have competing uses in the production of food and animal feed. In a step toward more sustainable industrial processes, we describe the conversion of P. pastoris into an autotroph that grows on CO2. By addition of eight heterologous genes and deletion of three native genes, we engineer the peroxisomal methanol-assimilation pathway of P. pastoris into a CO2-fixation pathway resembling the Calvin-Benson-Bassham cycle, the predominant natural CO2-fixation pathway. The resulting strain can grow continuously with CO2 as a sole carbon source at a µmax of 0.008 h-1. The specific growth rate was further improved to 0.018 h-1 by adaptive laboratory evolution. This engineered P. pastoris strain may promote sustainability by sequestering the greenhouse gas CO2, and by avoiding consumption of an organic feedstock with alternative uses in food production.