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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users

Exsolution Catalysts—Increasing Metal Efficiency

Exsolution catalysts are perovskite oxide-based materials that can exsolve catalytically active dopant elements as nanoparticles covering the surface, while the perovskite backbone can act as a stable support material. Thus, under proper conditions, a highly catalytically active and stable catalyst surface can be achieved. For many catalytic materials, precious metals or non-abundant elements play a key role in high catalytic activity. As these elements are often expensive or their supply is ecologically and ethically problematic, the replacement, or at the least reduction in the necessary amount used, is a common aim of current research. One strategy to do so is utilizing exsolution catalysts, as the active elements can be very selectively exsolved, and hence only very small doping amounts are sufficient for excellent results. This approach enables catalyst design with very high active metal efficiency

Quantitative Metabolite Profiling Utilizing Parallel Column Analysis for Simultaneous Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Separations Combined with Tandem Mass Spectrometry

In this work, a fully automated parallel LC column method was established in order to perform orthogonal hydrophilic interaction chromatography (HILIC) and reversed-phase (RPLC) chromatography within one analytical run for targeted quantitative mass spectrometric determination of metabolites from central carbon metabolism. In this way, the analytical throughput could be significantly improved compared to previously established dual separation work flows involving two separate analytical runs. Two sample aliquots were simultaneously injected onto a dual column setup columns using a ten-port valve, and parallel separations were carried out. Sub 2 μm particle size stationary phases were employed for both separation methods. HILIC and RPLC eluents were combined post column followed by ESI-MS/MS detection. The orthogonal separations were optimized, aiming at an overall separation with 2 retention time segments, while reversed-phase separation was accomplished within 5.5 min; metabolites on the HILIC phase were retained for a minimum time of 6 min. The overall run time was 15 min. The setup was applied to the quantification of 30 primary intercellular metabolites, including amino acids, organic acids, and nucleotides employing internal standardization by a fully ¹³C-labeled yeast extract. The comparison with HILIC–MS/MS and RPLC–MS/MS in separate analytical runs revealed that an excellent analytical performance was achieved by the parallel LC column method. The experimental repeatability (<i>N</i> = 5) was on average <5% (only for 2 compounds >5%). Moreover, limits of detection for the new approach ranging from 0.002–15 μM were in a good agreement with ones obtained in separate HILIC–MS/MS and RPLC–MS/MS runs (ranging from 0.01–44 μM)

In Situ Growth of Exsolved Nanoparticles under Varying rWGS Reaction Conditions&mdash;A Catalysis and Near Ambient Pressure-XPS Study

Perovskite-type oxides are highly flexible materials that show properties that are beneficial for application in reverse water-gas shift processes (rWGS). Due to their stable nature, the ability to incorporate catalytically active dopants in their lattice structure, and the corresponding feature of nanoparticle exsolution, they are promising candidates for a materials design approach. On an industrial level, the rWGS has proven to be an excellent choice for the efficient utilisation of CO2 as an abundant and renewable carbon source, reflected by the current research on novel and improved catalyst materials. In the current study, a correlation between rWGS reaction environments (CO2 to H2 ratios and temperature), surface morphology, and catalytic activity of three perovskite catalysts (Nd0.6Ca0.4Fe0.9Co0.1O3-&delta;, Nd0.6Ca0.4Fe0.97Co0.03O3-&delta;, and Nd0.6Ca0.4Fe0.97Ni0.03O3-&delta;) is investigated, combining catalytic measurements with SEM and NAP-XPS. The materials were found to react dynamically to the conditions showing both activation due to in situ nanoparticle exsolution and deactivation via CaCO3 formation. This phenomenon could be influenced by choice of material and conditions: less reductive conditions (larger CO2 to H2 or lower temperature) lead to smaller exsolved particles and reduced carbonate formation. However, the B-site doping was also important; only with 10% Co-doping, a predominant activation could be achieved

Doped metal clusters as bimetallic AuCo nanocatalysts: insights into structural dynamics and correlation with catalytic activity by in situ spectroscopy.

Acknowledgements: The authors acknowledge the support by the Austrian Science Fund (FWF) via grant Elise Richter (V831-N). ALBA synchrotron is acknowledged for the XAFS experiments at the CLAESS beamline (2017092492) with the collaboration of ALBA staff. Support during the experiments by Vera Truttmann, Clara Garcia and Stephan Pollitt are acknowledged.Co-doped Au25 nanoclusters with different numbers of doping atoms were synthesized and supported on CeO2. The catalytic properties were studied in the CO oxidation reaction. In all cases, an enhancement in catalytic activity was observed compared to the pure Au25 nanocluster catalyst. Interestingly, a different catalytic performance was obtained depending on the number of Co atoms within the cluster. This was related to the mobility of atoms within the cluster's structure under pretreatment and reaction conditions, resulting in active CoAu nanoalloy sites. The evolution of the doped Au clusters into nanoalloys with well-distributed Co atoms within the Au cluster structure was revealed by combined XAFS, DRIFTS, and XPS studies. Overall, these studies contribute to a better understanding of the dynamics of doped nanoclusters on supports upon pretreatment and reaction, which is key information for the future development and application of bimetallic nanocluster (nanoalloy) catalysts

Perovskite-Type Oxide Catalysts in CO2 Utilization: A Principal Study of Novel Cu-Doped Perovskites for Methanol Synthesis

Six different perovskite-type oxides were investigated with respect to their ability for methanol synthesis via H2 and CO2: Fe-, Mn-, and Ti-based perovskites were prepared with and without Cu doping. For assessment, the catalysts were subjected to preliminary tests at atmospheric pressure to evaluate their ability to activate CO2. Additional catalytic tests with the doped versions of each catalyst type were carried out in a pressured reactor at 21 bar. After the measurements, the catalysts were characterized with X-ray diffraction (XRD) and scanning electron microscopy (SEM). All catalysts were able to produce methanol in the pressure tests. CO2 conversions between 14% and 23% were reached at 400 &deg;C, with the highest methanol selectivity at the lower temperature of 250 &deg;C. The combination of XRD and SEM revealed that the Fe-based and Ti-based perovskites were stable under reaction conditions and that catalytically highly active and stable nanoparticles had formed. The minor formation of CaCO3, which is a deactivating phase, was observed for one catalyst. These nanoparticles showed resistance to coking and sintering. However, the yield and selectivity for methanol need to be improved via the further tailoring of the perovskite composition

Additional file 3: of Systems-level organization of yeast methylotrophic lifestyle

Enrichment of the peroxisomal marker protein Pex3p in the peroxisomal fraction. (PDF 271 kb

Additional file 1: of Systems-level organization of yeast methylotrophic lifestyle

Transcriptomic, proteomic, and metabolomic regulation of P. pastoris during methylotrophic growth. Containing the following eight sheets: Summary Omics Data: number of significantly regulated genes, proteins or metabolites (e.g. “up” refers to up-regulation in methanol/glycerol compared to glucose). Transcriptomics and proteomics: Average fold changes and P values of transcriptomics and proteomics comparing P. pastoris cultivated with methanol/glycerol or glucose as carbon source in chemostat. Average values derive from three biological replicates per condition. Metabolomics: Average fold changes and P values of metabolomics measurements comparing P. pastoris cultivated with methanol/glycerol or glucose as carbon source in chemostat cultivations. Average values derive from three biological replicates per condition. Co-regulation (related to Fig. 1 in the text): Regulation of the 575 gene-protein pairs with transcriptomics and proteomics data available and assignment to regulatory groups. Central carbon metabolism (related to Fig. 4 in the text): Average fold changes and P values of transcriptomics, proteomics, and metabolomics measurement depicted in Fig. 4. Amino acid metabolism (related to Fig. 6 in the text): Average fold changes and P values of transcriptomics, proteomics, and metabolomics measurement depicted in Fig. 6. Vitamin biosynthesis (related to Fig. 7 in the text): Average fold changes and P values of transcriptomics, proteomics, and metabolomics measurement depicted in Fig. 7. Peroxisomal gene regulation: Average fold changes and P values of transcriptomics and proteomics for all mentioned peroxisomal genes. Average values derive from three biological replicates per condition. (XLSX 2348 kb