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Occurrence and expression of tricarboxylate synthases in Ralstonia eutropha

  • Applied Microbial and Cell Physiology
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Abstract

2-Methylcitrate synthase (2-MCS1) and citrate synthase (CS) of Ralstonia eutropha strain H16 were separated by affinity chromatography and analyzed for their substrate specificities. 2-MCS1 used not only the primary substrate propionyl-CoA but also acetyl-CoA and, at a low rate, even butyryl-CoA and valeryl-CoA for condensation with oxaloacetate. The K M values for propionyl-CoA and acetyl-CoA were 0.061 or 0.35 mM, respectively. This enzyme is therefore a competitor for acetyl-CoA during biosynthesis of poly(3-hydroxybutyrate) (PHB) and has to be taken into account if metabolic fluxes are calculated for PHB biosynthesis. In contrast, CS could not use propionyl-CoA as a substrate. The gene-encoding CS (cisY) of R. eutropha was cloned and encodes for a protein consisting of 433 amino acids with a calculated molecular weight of 48,600 Da; it is not truncated in the N-terminal region. Furthermore, a gene encoding a second functionally active 2-methylcitrate synthase (2-MCS2, prpC2) was identified in the genome of R. eutropha. The latter was localized in a gene cluster with genes for an NAD(H)-dependent malate dehydrogenase and a putative citrate lyase. RT-PCR analysis of R. eutropha growing on different carbon sources revealed the transcription of prpC2. In addition, cells of recombinant Escherichia coli strains harboring prpC2 of R. eutropha exhibited high 2-MCS activity of 0.544 U mg−1. A prpC2 knockout mutant of R. eutropha exhibited an identical phenotype as the wild type if grown on different media. 2-MCS2 seems to be dispensable, and a function could not be revealed for this enzyme.

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Acknowledgements

We thank Katja Kemper for excellent technical assistance.

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  1. Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, 48149, Münster, Germany

    Christian Ewering, Christian O. Brämer, Nadine Bruland, Axel Bethke & Alexander Steinbüchel

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  1. Christian Ewering
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Correspondence to Alexander Steinbüchel.

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Ewering, C., Brämer, C.O., Bruland, N. et al. Occurrence and expression of tricarboxylate synthases in Ralstonia eutropha . Appl Microbiol Biotechnol 71, 80–89 (2006). https://doi.org/10.1007/s00253-005-0099-2

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