Novel intracellular 3-hydroxybutyrate-oligomer hydrolase in Wautersia eutropha H16

Abstract

Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.

FIG. 1.

Comparison of PhaZc amino acid sequences. A BLAST search using an intracellular 3HB-oligomer hydrolase amino acid sequence of Acidovorax sp. strain SA1 revealed several putative 3HB-oligomer hydrolases from PHB-accumulating bacteria. (A) Results of a multiple alignment using the Clustal W program (1.8.2). The candidate catalytic center is indicated by boldface type, and the lipase box is underlined. Asterisks indicate residues that are conserved in all proteins; colons indicate substitution of amino acid residues with very similar residues; and periods indicate substitution of amino acid residues with residues that are slightly less similar. (B) Phylogenetic tree. The cluster analysis was based on the neighbor-joining method. The numbers at the nodes are percentages of 1,000 bootstrap resamplings (only values greater than 50% are shown). The following proteins were included in the analyses: 3HB-oligomer hydrolase from Acidovorax sp. strain SA1 (SA1), predicted hydrolases from W. metallidurans (Wmet), R. solanacearum (Rsol), R. rubrum (Rrub), and R. sphaeroides (Rsph), and PhaZc from W. eutropha cloned in this study (Weut). The accession numbers of these proteins are AB044565, ZP_00276649, NP_519695, ZP_00268816, ZP_00007013, and AB180691, respectively.

FIG. 2.

SDS-PAGE analysis of purified PhaZc from E. coli harboring pE3ReZc. E. coli overexpressing phaZc was sonicated and centrifuged, and the soluble crude extract (14 μg) (lane 1) was purified on a hydrophobic column (2 μg) (lane 2) and an anion-exchange column (1 μg) (lane 3). The arrow indicates the position of PhaZc. Lane M contained molecular mass markers.

FIG. 3.

Degradation of artificial amorphous PHB granules by PhaZc. The 3HB released during PHB degradation was measured enzymatically. Symbols: ×, no PhaZc; ▴, 0.46 μg of PhaZc; ⧫, 1.2 μg of PhaZc; •, 4.6 μg of PhaZc; ○, 4.6 μg of boiled PhaZc (100°C, 5 min). The error bars indicate standard deviations of the means based on three independent measurements.

FIG. 4.

Subcellular localization of PhaZc determined by using a sucrose density gradient. Sonicates from PHB-rich cells were fractionated by ultracentrifugation in a sucrose gradient (1 to 2 M; total volume, 11 ml). Each fraction (1.1 ml) was collected and analyzed by Western blotting and immunostaining with antiserum against PhaZc, PhaZ1, or 3HB dehydrogenase (3HBDH); 100% corresponded to PHB and protein concentrations of 65 and 3.7 mg/ml, respectively.

FIG. 5.

Growth and accumulation of PHB in various W. eutropha phaZ deletion mutants in the nutrient-rich medium and the PHB accumulation medium. (A and B) Nutrient-rich medium. (C and D) PHB accumulation medium (mineral salt medium containing 2% [wt/vol] fructose and 0.1% [wt/vol] ammonium sulfate). (A and C) Cell dry weight. (B and D) PHB content. Symbols: ○, wild type; ▵, TK0120 (ΔphaZc mutant); □, OH1 (ΔphaZb mutant); ⋄, H16DZbc1 (ΔphaZb ΔphaZc mutant). The data are means for three independent measurements. The error bars indicate standard deviations.