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Graz, Austria

Sreckovic I.,Medical University of Graz | Birner-Gruenberger R.,Medical University of Graz | Birner-Gruenberger R.,Austrian Center of Industrial Biotechnology | Birner-Gruenberger R.,Omics Center Graz | And 10 more authors.
Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids

Gestational diabetes mellitus (GDM) is related to neonatal macrosomia and an increased risk of vascular events. We hypothesized that GDM exerts qualitative effects on neonatal high-density lipoprotein (HDL). HDL was isolated from control (n = 11) and GDM maternal/neonatal donors (n = 9) and subjected to shotgun proteomics. Differences in HDL mobility were assessed by FPLC and native gel-electrophoresis. Paraoxonase (PON1) activity, cholesterol ester-transfer protein (CETP) mass and activity, phospholipid, triglyceride and cholesterol concentrations were quantified with commercial kits. Total anti-oxidative capacity and cholesterol efflux capability of HDLs were measured. Four proteins involved in lipid metabolism, inflammation and innate immunity were differentially expressed between controls and GDM neonates. ApoM (decreased, p < 0.05) and SAA1 (increased, p < 0.05) showed the same differences on both, maternal and neonatal GDM HDL. Lower PON1 protein expression was corroborated by lower activity (p < 0.05) which in turn was associated with attenuated anti-oxidant capacity of GDM HDL. Protein changes were accompanied by increased levels of triglycerides and decreased levels of cholesterol esters, respectively. The observed differences in GDM HDL lipid moiety may be related to CETP mass and activity alterations. The rate of cholesterol efflux from term trophoblasts to maternal and from placental endothelial cells to neonatal GDM HDL was impaired (p < 0.05). In conclusion, GDM causes changes in HDL composition and is intimately associated with impaired cholesterol efflux capability as well as diminished anti-oxidative particle properties. Remodeling of neonatal GDM HDL in utero supports the hypothesis that maternal conditions in pregnancy impact neonatal lipoprotein metabolism. © 2014 Published by Elsevier B.V. Source

Vogl T.,University of Graz | Thallinger G.G.,University of Graz | Thallinger G.G.,Omics Center Graz | Thallinger G.G.,Austrian Center of Industrial Biotechnology | And 5 more authors.
New Biotechnology

Membrane proteins are the largest group of human drug targets and are also used as biocatalysts. However, due to their complexity, efficient expression remains a bottleneck for high level production. In recent years, the methylotrophic yeast Pichia pastoris has emerged as one of the most commonly used expression systems for membrane protein production.Here, we have analysed the transcriptomes of P. pastoris strains producing different classes of membrane proteins (mitochondrial, ER/Golgi and plasma membrane localized) to understand the cellular response and to identify targets to engineer P. pastoris towards an improved chassis for membrane protein production.Microarray experiments revealed varying transcriptional responses depending on the enzymatic activity, subcellular localization and physiological role of the membrane proteins. While an alternative oxidase evoked primarily a response within the mitochondria, the overexpression of transporters entering the secretory pathway had a wide effect on lipid metabolism and induced the upregulation of the UPR (unfolded protein response) transcription factor Hac1p. Coexpression of P. pastoris endogenous HAC1 increased the levels of ER-resident membrane proteins 1.5- to 2.1-fold. Subsequent transcriptome analysis of HAC1 coexpression revealed an upregulation of the folding machinery correlating with an expansion of the ER membrane capacity, thus boosting membrane protein production. Hence, our study has helped to elucidate the cellular response of P. pastoris to the expression of different classes of membrane proteins and led specifically to new insights into the effect of PpHac1p on membrane proteins entering the secretory pathway. © 2014 Elsevier B.V.. Source

Knittelfelder O.L.,University of Graz | Weberhofer B.P.,University of Graz | Eichmann T.O.,University of Graz | Kohlwein S.D.,University of Graz | And 2 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

A new UPLC-based untargeted lipidomic approach using a qTOF hybrid mass spectrometer is introduced. The applied binary gradient enables separations of lipid species including constitutional isomeric compounds and low abundant lipid classes such as phosphatidic acid (PA). Addition of phosphoric acid to the solvents improves peak shapes for acidic phospholipids. MSE scans allow simultaneous acquisition of full scan data and collision induced fragmentation to improve identification of lipid classes and to obtain structural information. The method was used to investigate the lipidome of yeast. © 2014 The Authors. Source

Klapper M.,University of Kiel | Findeis D.,TU Braunschweig | Koefeler H.,University of Graz | Koefeler H.,Omics Center Graz | Doring F.,University of Kiel
Genes and Nutrition

Background: Almost all animals adapt to dietary restriction through alternative life history traits that affect their growth, reproduction, and survival. Economized management of fat stores is a prevalent type of such adaptations. Because one-carbon metabolism is a critical gauge of food availability, in this study, we used Caenorhabditis elegans to test whether the methyl group donor choline regulates adaptive responses to dietary restriction. We used a modest dietary restriction regimen that prolonged the fecund period without reducing the lifetime production of progeny, which is the best measure of fitness. Results: We found that dietary supplementation with choline abrogate the dietary restriction-induced prolongation of the reproductive period as well as the accumulation and delayed depletion of large lipid droplets and whole-fat stores and increased the survival rate in the cold. By contrast, the life span-prolonging effect of dietary restriction is not affected by choline. Moreover, we found that dietary restriction led to the enlargement of lipid droplets within embryos and enhancement of the cold tolerance of the progeny of dietarily restricted mothers. Both of these transgenerational responses to maternal dietary restriction were abrogated by exposing the parental generation to choline. Conclusions: In conclusion, supplementation with the methyl group donor choline abrogates distinct responses to dietary restriction related to reproduction, utilization of fat stored in large lipid droplets, cold tolerance, and thrifty phenotypes in C. elegans. © 2016 Klapper et al. Source

Kavscek M.,University of Graz | Bhutada G.,University of Graz | Madl T.,Medical University of Graz | Madl T.,Omics Center Graz | And 3 more authors.
BMC Systems Biology

Background: Yarrowia lipolytica is a non-conventional yeast that is extensively investigated for its ability to excrete citrate or to accumulate large amounts of storage lipids, which is of great significance for single cell oil production. Both traits are thus of interest for basic research as well as for biotechnological applications but they typically occur simultaneously thus lowering the respective yields. Therefore, engineering of strains with high lipid content relies on novel concepts such as computational simulation to better understand the two competing processes and to eliminate citrate excretion. Results: Using a genome-scale model (GSM) of baker's yeast as a scaffold, we reconstructed the metabolic network of Y. lipolytica and optimized it for use in flux balance analysis (FBA), with the aim to simulate growth and lipid production phases of this yeast. We validated our model and found the predictions of the growth behavior of Y. lipolytica in excellent agreement with experimental data. Based on these data, we successfully designed a fed-batch strategy to avoid citrate excretion during the lipid production phase. Further analysis of the network suggested that the oxygen demand of Y. lipolytica is reduced upon induction of lipid synthesis. According to this finding we hypothesized that a reduced aeration rate might induce lipid accumulation. This prediction was indeed confirmed experimentally. In a fermentation combining these two strategies lipid content of the biomass was increased by 80 %, and lipid yield was improved more than four-fold, compared to standard conditions. Conclusions: Genome scale network reconstructions provide a powerful tool to predict the effects of genetic modifications and the metabolic response to environmental conditions. The high accuracy and the predictive value of a newly reconstructed GSM of Y. lipolytica to optimize growth conditions for lipid accumulation are demonstrated. Based on these findings, further strategies for engineering Y. lipolytica towards higher efficiency in single cell oil production are discussed. © 2015 Kavšček et al. Source

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