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Characterization of membrane-bound dehydrogenases from Gluconobacter oxydans 621H via whole-cell activity assays using multideletion strains

  • Applied genetics and molecular biotechnology
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Abstract

Gluconobacter oxydans, like all acetic acid bacteria, has several membrane-bound dehydrogenases, which oxidize a multitude of alcohols and polyols in a stereo- and regio-selective manner. Many membrane-bound dehydrogenases have been purified from various acetic acid bacteria, but in most cases without reporting associated sequence information. We constructed clean deletions of all membrane-bound dehydrogenases in G. oxydans 621H and investigated the resulting changes in carbon utilization and physiology of the organism during growth on fructose, mannitol, and glucose. Furthermore, we studied the substrate oxidation spectra of a set of strains where the membrane-bound dehydrogenases were consecutively deleted using a newly developed whole-cell 2,6-dichlorophenolindophenol (DCPIP) activity assay in microtiter plates. This allowed a detailed and comprehensive in vivo characterization of each membrane-bound dehydrogenase in terms of substrate specificity. The assays revealed that general rules can be established for some of the enzymes and extended the known substrate spectra of some enzymes. It was also possible to assign proteins whose purification and characterization had been reported previously, to their corresponding genes. Our data demonstrate that there are less membrane-bound dehydrogenases in G. oxydans 621H than expected and that the deletion of all of them is not lethal for the organism.

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Acknowledgments

We thank the Bundesministerium für Bildung und Forschung (BMBF) for funding this work in the framework of the GenoMik-Transfer initiative (FKZ: 0315632C).

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  1. Lehrstuhl für Mikrobiologie, Technische Universität München, Emil-Ramann-Strasse 4, 85354, Freising, Germany

    Björn Peters, Markus Mientus, David Kostner, Anja Junker, Wolfgang Liebl & Armin Ehrenreich

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  1. Björn Peters
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  2. Markus Mientus
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  3. David Kostner
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  4. Anja Junker
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  5. Wolfgang Liebl
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  6. Armin Ehrenreich
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Correspondence to Armin Ehrenreich.

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Peters, B., Mientus, M., Kostner, D. et al. Characterization of membrane-bound dehydrogenases from Gluconobacter oxydans 621H via whole-cell activity assays using multideletion strains. Appl Microbiol Biotechnol 97, 6397–6412 (2013). https://doi.org/10.1007/s00253-013-4824-y

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  • DOI: https://doi.org/10.1007/s00253-013-4824-y

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