Open Access Highly Accessed Research article

A multi-level study of recombinant Pichia pastoris in different oxygen conditions

Kristin Baumann1, Marc Carnicer1, Martin Dragosits2, Alexandra B Graf23, Johannes Stadlmann4, Paula Jouhten5, Hannu Maaheimo5, Brigitte Gasser2, Joan Albiol1, Diethard Mattanovich23 and Pau Ferrer1*

Author Affiliations

1 Department of Chemical Engineering, Autonomous University of Barcelona, Spain

2 Institute of Applied Microbiology, Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria

3 School of Bioengineering, University of Applied Sciences, FH Campus Vienna, Austria

4 Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria

5 VTT Technical Research Centre of Finland, Espoo, Finland

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BMC Systems Biology 2010, 4:141  doi:10.1186/1752-0509-4-141

Published: 22 October 2010

Additional files

Additional file 1:

Design 2D DIGE Gels. An example of the experimental design for the acquisition of statistical data on differences between samples taken from normoxic (21%), oxygen-limiting (11%) and hypoxic (8%) setpoints. Replica of 2 independent experiments (F1 or F2; F = fermentation) were labelled with either Cy5 or Cy3 (GE Healthcare). A pool of all samples served as reference and was labelled with Cy2 (= pooled standard).

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Additional file 2:

2D DIGE Gels. Representative gel image from a 2D gel electrophoresis experiment with proteins obtained from the Fab-expressing strain. We identified 45 out of 81 proteins with a different expression pattern when comparing high and low oxygen experiments. Green spots show proteins downregulated and pink ones show those upregulated under hypoxia.

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Additional file 3:

Identified protein spots. List of the 45 identified proteins with different abundances comparing hypoxic (8) and normoxic (21) conditions in the P. pastoris expressing and control strain. Proteins were identified by MALDI-TOF MS and grouped into 6 different biological processes. The protein name, short name and accession number, theoretical Mw and pI are reported together with the percentage of peptide coverage and number of identified peptides. Average ratios and 1-ANOVA (DeCyder) are given and only not indicated where no spot could be matched. p.i. = previously identified on other 2D gels

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Additional file 4:

Relative abundances of intact carbon fragments in proteinogenic amino acids. Relative abundances of intact C2 and C3 fragments (f-values) in proteinogenic amino acids describing the conservation of carbon chain fragments in P. pastoris Fab-producing and control strains growing in glucose-limited chemostats at a D = 0.1 h-1 in different oxygenation conditions.

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Additional file 5:

Metabolic fluxes Metabolic fluxes in the central carbon metabolism of P. pastoris Fab-producing and control strain in glucose-limited chemostats at a D = 0.1 h-1 in different oxygenation conditions. The standard deviations of each net flux are given.

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Additional file 6:

qRT-PCR primer sequences used in this study. Table showing the primer sequences of the genes analyzed by qRT-PCR and characteristics of the corresponding amplicons. Calculated copy numbers and the dilution factors in order to obtain 109 copies μl-1 are indicated (see main text for explanation).

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Additional file 7:

Metabolic network model of the central carbon metabolism of P. pastoris. Bioreaction network model of the central carbon metabolism of P. pastoris used in the 13C-metabolic flux analysis for the determination of net fluxes under the different oxygenation conditions. Fluxes are represented as net fluxes and the directions of the arrows indicate the directions of the positive net fluxes. The metabolites consumed or produced by extracellular fluxes (shown as dashed arrows) are denoted with (E).

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Additional file 8:

Stoichiometric model of the central carbon metabolism of P. pastoris. Reactions in the stoichiometric model of the central carbon metabolism of P. pastoris applied in the 13C-MFA determination of the metabolic fluxes under different oxygenation conditions; it also includes anabolic reactions from metabolic intermediates to biosynthesis, transport reactions across the mitochondrial membrane and uptake and excretion reactions. Note that O2, CO2, energy and redox cofactor mass balances were not included in the mass balance constraints for 13C-MFA.

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