Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene fromRalstonia eutrophaH16 and Characterization of the Gene Product

Abstract

An intracellular poly[d(−)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned fromRalstonia eutrophaH16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract ofEscherichia colicontaining the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomericd(−)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed inR. eutrophacells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant ofR. eutrophawhosephaZgene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the clonedphaZgene encodes an intracellular PHB depolymerase inR. eutropha.