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Scale-down effect on the extracellular proteome of Escherichia coli: correlation with membrane permeability and modulation according to substrate heterogeneities

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Abstract

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane permeability and the synthesis of several outer-membrane components allowing to cope with substrate limitation commonly found in high cell density culture. A comparative analysis of protein leakage has thus been performed in well-mixed bioreactors and in scale-down devices. The extracellular proteome of E.coli has been investigated by 2D-gel electrophoresis and identified by subsequent MALDI-TOF analysis. On 110 picked spots, 67 proteins have been identified and the sub-localisation and the molecular function of these proteins have been determined. A majority of the extracellular proteome was composed of outer-membrane and periplasmic proteins (64 %) confirming the fact that leakage is involved in high cell density cultures. About 50 % of this extracellular proteome was composed of transport and binding proteins. Furthermore, the more abundant spots on the gel corresponded to porin proteins and periplasmic transporters. In particular, the OmpC porin was found to be very abundant. Moreover, the scale-down effect on this extracellular proteome has been investigated by two-dimensional differential in-gel electrophoresis analysis (2D-DIGE), and significant differences have been observed by comparison with culture carried out in well-mixed systems. Indeed, since substrate limitation signal is alleviated in this kind of apparatus, cell permeability was lowered as shown by flow cytometry. In scale-down conditions, protein leakage was thus less abundant.

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Acknowledgments

AB is recipient of a PhD FRIA grant provided by the Belgian Fund for Scientific Research (FRS-FNRS).

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Authors and Affiliations

  1. Université de Liège, Gembloux Agro-Bio Tech, Unité de Bio-industries/CWBI, Passage des Déportés 2, 5030, Gembloux, Belgium

    Alison Brognaux, Philippe Thonart & Frank Delvigne

  2. Fond de la recherche scientifique (FRS-FNRS), Rue d’Egmont 5, 1000, Brussels, Belgium

    Alison Brognaux

  3. Université de Liège, Gembloux Agro-Bio Tech, Unité d’entomologie fonctionnelle et évolutive, Passage des Déportés 2, 5030, Gembloux, Belgium

    Frédéric Francis

  4. Université de Liège, Gembloux Agro-Bio Tech, Unité de biologie cellulaire et moléculaire, Passage des Déportés 2, 5030, Gembloux, Belgium

    Jean-Claude Twizere

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  1. Alison Brognaux
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  2. Frédéric Francis
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Correspondence to Alison Brognaux.

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Brognaux, A., Francis, F., Twizere, JC. et al. Scale-down effect on the extracellular proteome of Escherichia coli: correlation with membrane permeability and modulation according to substrate heterogeneities. Bioprocess Biosyst Eng 37, 1469–1485 (2014). https://doi.org/10.1007/s00449-013-1119-8

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