Poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 is involved in mobilization of accumulated PHB in Ralstonia eutropha H16

Abstract

The recently finished genome sequence of Ralstonia eutropha H16 harbors nine genes that are thought to encode functions for intracellular depolymerization (mobilization) of storage poly(3-hydroxybutyrate) (PHB). Based on amino acid similarities, the gene products belong to four classes (PhaZa1 to PhaZa5, PhaZb, PhaZc, and PhaZd1/PhaZd2). However, convincing direct evidence for the in vivo roles of the gene products is poor. In this study, we selected four candidate genes (phaZa1, phaZb, phaZc, and phaZd1) representing the four classes and investigated the physiological function of the gene products (i) with recombinant Escherichia coli strains and (ii) with R. eutropha null mutants. Evidence for weak but significant PHB depolymerase activity was obtained only for PhaZa1. The physiological roles of the other potential PHB depolymerases remain uncertain.

FIG. 1.

Growth (turbidity) and glucose consumption (A); cellular dry weight (CDW)/liter and pH profile (B); and secretion of 3HB (C) with NaOH treatment for the determination of total 3HB concentration (dotted lines) or without NaOH treatment (solid lines) by E. coli XL1-Blue harboring different combinations of R. eutropha pha genes (Table 1). Growth was measured in R medium with 20 g/liter glucose without pH control as described in references and . Three independent experiments have been performed. A typical result is shown.

FIG. 2.

Secretion of 3HB by washed cells of recombinant E. coli BL21 harboring different combination of pha genes (Table 1). Bacteria were grown in the presence of rhamnose and IPTG as described in the text. Washed cells were incubated in buffer (100 mM potassium phosphate [pH 7]) at 37°C. Cell-free samples taken at the time points shown were assayed for amounts of 3HB released, by 3HB dehydrogenase assay. Three independent experiments have been performed. A typical result is shown.

FIG. 3.

Secretion of 3HB by washed cells of R. eutropha H16 and phaZ null mutants as indicated. Bacteria were grown in mineral salts medium (35) with gluconate. Washed cells were incubated in buffer (100 mM potassium phosphate [pH 7]) at 30°C. Cell-free samples taken at the time points shown were assayed for the amounts of 3HB released, by 3HB dehydrogenase assay.