Novel functions associated with poly(3-hydroxybutyrate) (PHB) granules

• Applicant: Professor Dr. Dieter Jendrossek
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 269261353

Comparative proteome analysis of four subcellular fractions revealed the presence of 14 (out of ca. 3000 identified) specifically PHB granule associated proteins (PGAPs) in Ralstonia eutropha H16. In addition to 10 previously described PGAPs four proteins were identified that have not been pulled together with PHB metabolism before: one is a hypothetical protein of unknown function, two are putative a/b-hydrolases with PHB synthase/depolymerase signatures and the fourth is a patatin-like phospholipase. In this project we will determine the function of these proteins. We hypothesize that the two a/b-hydrolases are novel PHB depolymerases that cleave PHB by thiolysis. The product, 3-hydroxybutyryl-CoA, could be used to generate ATP via phospho-transacetylase (Pta1) and acetate kinase (AckA), the genes of which were expressed and identified in an operon together with one of the two new a/b-hydrolases. The phospholipase could be responsible for either cleaving phospholipids from the PHB granule surface or for synthesis of phospholipids depending on the in vivo direction of catalysis on the hydrophobic PHB surface. Determination of the phenotypes of deletions of the four genes, biochemical analysis of the purified proteins and of isolated PHB granules from wt and mutants, together with localization of phospholipids by GFP fusions with specific phospholipid-binding proteins in wt and mutant strains will be used to identify the functions of the new proteins. This project will answer the 50 years old question on the absence or presence of a phospholipid layer in PHB granules in vivo, and we hope to confirm the postulated formation of ATP from PHB via thiolysis and substrate level phosphorylation, a not yet described function of PHB organelles.

• DFG Programme: Research Grants