Cloning of an intracellular poly[D(-)-3-hydroxybutyrate] depolymerase gene from Ralstonia eutropha H16 and characterization of the gene product

An intracellular poly[D(-)3-hydrolrybutyrate] (PHB) depolymerase gene (phaZ) has been cloned from Ralstonia eutropha H16 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 of Escherichia coli containing the PWB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric D(-)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 in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutropha whose phaZ gene 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 cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.