caa a22 4500 605505497 CHVBK 20210128100622.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6972-8 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6972-8 SdhE-dependent formation of a functional Acetobacter pasteurianus succinate dehydrogenase in Gluconobacter oxydans —a first step toward a complete tricarboxylic acid cycle [Elektronische Daten] [Ines Kiefler, Stephanie Bringer, Michael Bott] The obligatory aerobic α-proteobacterium Gluconobacter oxydans 621H possesses an unusual metabolism in which the majority of the carbohydrate substrates are incompletely oxidized in the periplasm and only a small fraction is metabolized in the cytoplasm. The cytoplasmic oxidation capabilities are limited due to an incomplete tricarboxylic acid (TCA) cycle caused by the lack of succinate dehydrogenase (Sdh) and succinyl-CoA synthetase. As a first step to test the consequences of a functional TCA cycle for growth, metabolism, and bioenergetics of G. oxydans, we attempted to establish a heterologous Sdh in this species. Expression of Acetobacter pasteurianus sdhCDAB in G. oxydans did not yieldan active succinate dehydrogenase. Co-expression of a putative sdhE gene from A. pasteurianus, which was assumed to encode an assembly factor for covalent attachment of flavin adenine dinucleotide (FAD) to SdhA, stimulated Sdh activity up to 400-fold to 4.0±0.4U (mg membrane protein)‒1. The succinate/oxygen reductase activity of membranes was 0.68±0.04U (mg membrane protein)‒1, indicating the formation of functional Sdh complex capable of transferring electrons from succinate to ubiquinone. A. pasteurianus SdhE could be functionally replaced by SdhE from the γ-proteobacterium Serratia sp. According to these results, the accessory protein SdhE was necessary and sufficient for heterologous synthesis of an active A. pasteurianus Sdh in G. oxydans. Studies with the Sdh-positive G. oxydans strain provided evidence for a limited functionality of the TCA cycle despite the absence of succinyl-CoA synthetase. Springer-Verlag Berlin Heidelberg, 2015 Gluconobacter oxydans nationallicence Citrate cycle nationallicence Succinate dehydrogenase nationallicence Kiefler Ines Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany aut Bringer Stephanie Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany aut Bott Michael Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 9147-9160 0175-7598 99:21<9147 2015 99 253 https://doi.org/10.1007/s00253-015-6972-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6972-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kiefler Ines Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany aut NATIONALLICENCE 700 1- Bringer Stephanie Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany aut NATIONALLICENCE 700 1- Bott Michael Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 9147-9160 0175-7598 99:21<9147 2015 99 253