Occurrence and expression of tricarboxylate synthases in Ralstonia eutropha

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 KM 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.

<i>Ralstonia eutropha</i> Strain H16 as Model Organism for PHA Metabolism and for Biotechnological Production of Technically Interesting Biopolymers

“…R. eutropha grows well on levulinic acid and degrades it via 4-hydroxyvaleryl-CoA to propionyl-CoA plus acetyl-CoA. Propionyl-CoA derived from levulinic acid, propionate or other carbon sources is then utilized via the 2-methylcitric acid cycle (2MCC) and not via the methyl-malonylCoA pathway [Brämer and Steinbüchel, 2001;Ewering et al, 2006a]. An engineered strain of R. eutropha , in which the 2-methyl-cis -aconitate hydratase (acnM) gene was disrupted by directed insertion of an additional copy of a 2-methylcitrate synthase gene (prpC) , could be utilized for production of high concentrations of 2-methylcitrate in the medium when cultivated with levulinic acid plus succinate as carbon sources [Ewering et al, 2006b].…”

Chicken feather hydrolysate as an inexpensive complex nitrogen source for PHA production byCupriavidus necatoron waste frying oils

“…Generally, only a small portion (less than 15% of carbon) of available propionyl‐CoA is incorporated into P(3HB‐ co ‐3HV) (Ewering et al . ). Taking into account the costs of 3HV precursors, the inefficiency of precursor incorporation into the copolymer negatively influences the economics of PHA production (Obruca et al .…”