MOESM1 of Biomarkers allow detection of nutrient limitations and respective supplementation for elimination in Pichia pastoris fed-batch cultures

Additional file 1: Figure S1. Mean expression values (microarray data) of the carboxypeptidase B gene during the methanol and glucose fed-batch process. Table S2. Description of biological function of marker genes adapted from Saccharomyces Genome Database (SGD). Table S3. qPCR primer sequences. Figure S4. Principal component analysis (variable factor map; F2 vs. F1) of the methanol fed-batch (1 h, 15 h, 27 h, 41 h, 53 h and 67 h after starting the methanol fed-batch) referred to the glycerol fed-batch (log2 fold change). Figure S5. Principal component analysis (variable factor map; F3 vs. F2) of the methanol fed-batch (1 h, 15 h, 27 h, 41 h, 53 h and 67 h after starting the methanol fed-batch) referred to the glycerol fed-batch (log2 fold change). Figure S6. Principal component analysis (variable factor map; F2 vs. F1) of the glucose fed-batch (1 h, 15 h, 27 h, 41 h, 53 h and 67 h after starting the glucose fed-batch) referred to the glycerol fed-batch (log2 fold change). Figure S7. Principal component analysis (variable factor map; F3 vs. F2) of the glucose fed-batch (1 h, 15 h, 27 h, 41 h, 53 h and 67 h after starting the glucose fed-batch) referred to the glycerol fed-batch (log2 fold change)

Gas Chromatography-Quadrupole Time-of-Flight Mass Spectrometry-Based Determination of Isotopologue and Tandem Mass Isotopomer Fractions of Primary Metabolites for ¹³C‑Metabolic Flux Analysis

For the first time an analytical work flow based on accurate mass gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOFMS) with chemical ionization for analysis providing a comprehensive picture of ¹³C distribution along the primary metabolism is elaborated. The method provides a powerful new toolbox for ¹³C-based metabolic flux analysis, which is an emerging strategy in metabolic engineering. In this field, stable isotope tracer experiments based on, for example, ¹³C are central for providing characteristic patterns of labeled metabolites, which in turn give insights into the regulation of metabolic pathway kinetics. The new method enables the analysis of isotopologue fractions of 42 free intracellular metabolites within biotechnological samples, while tandem mass isotopomer information is also accessible for a large number of analytes. Hence, the method outperforms previous approaches in terms of metabolite coverage, while also providing rich isotopomer information for a significant number of key metabolites. Moreover, the established work flow includes novel evaluation routines correcting for isotope interference of naturally distributed elements, which is crucial following derivatization of metabolites. Method validation in terms of trueness, precision, and limits of detection was performed, showing excellent analytical figures of merit with an overall maximum bias of 5.8%, very high precision for isotopologue and tandem mass isotopomer fractions representing >10% of total abundance, and absolute limits of detection in the femtomole range. The suitability of the developed method is demonstrated on a flux experiment of <i>Pichia pastoris</i> employing two different tracers, i.e., 1,6¹³C₂-glucose and uniformly labeled ¹³C-glucose

Growth curves of wild type and L-ascorbic acid producing yeasts under oxidative stress.

<p>Kinetics of growth of wild type and engineered strains GRF18U (left panels) and BY4742 (right panels) as inoculated in minimal glucose media without H₂O₂ (3A and 3B) or in presence of H₂O₂ 3.5 mM (3C and 3D). • GRF18U wild type; ▪ GRF18U[<i>ScALO AtLGDH AtME AtMIP</i>]; ▴ GRF18U[<i>ScALO AtLGDH AtME AtMIP RnFGT</i>]; ○ BY4742 wild type; □ BY4742[<i>ScALO AtLGDH AtME AtMIP</i>]; ▵ BY4742[<i>ScALO AtLGDH AtME AtMIP RnFGT</i>].</p

List of expression plasmids constructed and used in this study*

<p>Abbreviations: Sc: <i>Saccharomyces cerevisiae</i>; At: <i>Arabidopsis thaliana</i>; Rn: <i>Rattus norvegicus</i>; Zb: <i>Zygosaccharomyces bailii</i>;</p><p>TPI: Triose Phosphate Isomerase</p><p><i>URA3, HIS3, LEU2, LYS2</i>: gene markers conferring growth to auxotrophic yeast strains in the absence of uracil, histidine, leucine and lysine, respectively.</p><p>Nat^R: cassette conferring resistance to nourseotricine.</p><p>Kan^R: cassette conferring resistance to Geneticin.</p><p>CEN and INT: centromeric and integrative plasmids, respectively.</p>*<p>a complete description of plasmids construction is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001092#s4" target="_blank">Materials and Methods</a></p

Flow cytometric analysis of wild type and vitamin C producing yeasts under oxidative stress.

<p>Panel 5A: schematic representation of the different subpopulations that can be observed in the following panels, where wild type (5B) and recombinant strains (5C and 5D) grown in minimal glucose medium added with H₂O₂ 3.0 mM were analyzed after DHR123 and PI staining (rodamine signal is reported in the abscissa and PI signal on the ordinate axes). Upper panels: GRF18U background. Lower panels: BY background. (B): wild type. (C): [<i>ScALO AtLGDH AtME AtMIP</i>] transformed cells. (D): [<i>ScALO AtLGDH AtME AtMIP RnFGT</i>] transformed cells</p

Conversion of D-Glucose into L-ascorbic acid (milligrams/liter/OD) by transformed <i>S. cerevisiae</i> GRF18U and BY4742 cells.

<p>All strains were grown on mineral medium (2% w/v glucose, 0.67% w/v YNB), starting with an initial OD660 of 0.05 for 18 h, when samples were taken and the concentration of L-ascorbic acid inside the cells was determined (GRF18U and BY4742 correspond to the parental strains transformed with the empty plasmids harboring in the productive strains the genes of the L-AA pathway). The control cells, as well as the cells expressing <i>ScALO1</i> and <i>AtLGDH</i> can not accumulate L-ascorbic acid starting from D-glucose, therefore measured values correspond to the endogenous erythro-ascorbic acid. The standard deviation bars correspond to the data obtained from independent clones, and from independent growth and antioxidant determinations. Please note the different scale of the ordinate axes in the two graphs.</p

List of yeast strains constructed and used in this study

<p>List of yeast strains constructed and used in this study</p

MOESM2 of Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris

Additional file 2. Table S4. Genome sequencing statistic

Monitoring of transcriptional regulation in under protein production conditions-4

<p><b>Copyright information:</b></p><p>Taken from "Monitoring of transcriptional regulation in under protein production conditions"</p><p>http://www.biomedcentral.com/1471-2164/8/179</p><p>BMC Genomics 2007;8():179-179.</p><p>Published online 19 Jun 2007</p><p>PMCID:PMC1919374.</p><p></p>steady state; Blue bars: 20 °C steady state. BiP: intracellular signals for the UPR marker BiP/Kar2p (detected with anti-Grp78/BiP specific IgG). HC: intracellular signals for Fab heavy chain (obtained with anti-h-Fab specific IgG); LC: intracellular signals for light chain (analyzed with anti-kappa light chain IgG)

Monitoring of transcriptional regulation in under protein production conditions-1

<p><b>Copyright information:</b></p><p>Taken from "Monitoring of transcriptional regulation in under protein production conditions"</p><p>http://www.biomedcentral.com/1471-2164/8/179</p><p>BMC Genomics 2007;8():179-179.</p><p>Published online 19 Jun 2007</p><p>PMCID:PMC1919374.</p><p></p>ndend up- or downregulatated genes are highlighted; B: Logratio of cultures producing 2F5 Fab under control of the GAP promoter compared to the wild type, in the same order as A. Red bars: change in transcript up > 2-fold; yellow bars: up > 1.5 fold; white bars: unchanged; blue bars: down > 1.5 fold