Elevated poly(3-hydroxybutyrate) synthesis in mutants of Ralstonia eutropha H16 defective in lipopolysaccharide biosynthesis

Abstract

Several independent transposon Tn5-induced mutants of Ralstonia eutropha H16 exhibited a poly(3-hydroxybutyric acid) (PHB) elevated phenotype and accumulated substantial amounts of PHB already in the exponential growth phase. The insertion loci of Tn5 in these six mutants were mapped in the genes hldA (twice), hldC (twice), rfaF2, and rfaF3, which are all involved in the synthesis of lipopolysaccharides (LPS), an important component of the outer membrane (OM) of Gram-negative bacteria. The generated defined deletion mutant ΔhldA confirmed the PHB elevated phenotype. According to the literature,such a truncated LPS may cause an increased permeability of the OM; thereby, the mutations may lead to a facilitated uptake of carbon source from the medium as exemplarily shown for gluconate and succinate. Thus, the ratio of carbon to nitrogen in the cell is increased. Proteome analyses revealed reinforcement of the Entner–Doudoroff pathway and of subsequent reactions that finally may lead to higher concentrations of acetyl-CoA in the cells. Due to the impaired synthesis of complete LPS, intermediates of LPS biosynthesis might be recycled by reactions yielding higher levels of NADPH and acetyl-CoA. Since the latter are precursors for synthesis of PHB, this could explain the elevated synthesis and accumulation of this polymer in case of the LPS mutants.