Cloning and Sequencing of the Poly (3-hydroxybutyrate)(PHB) Synthase Gene from Purple Non-Sulfur Bacteria Rhodospirillum centenum and Expression of the Gene in Escherichia coli

A 10-kb genomic fragment was isolated from Rhodospirillum centenum by Southern-hybridization and colony-hybridization, using a probe amplified by PCR with oligo-nucleotide primers constructed from a sequence conserved in poly (3-hydro xybutyrate)(PHB) synthase genes. After subcloning of an approximately 3-kb fragment (Smal-EcoRV) that caused the production of PHB in Escherichia coli in the presence of 13-ketothiolase (phbA), an acetoacety-CoA reductase (phbB) gene from Ralstonia eutropha H16 was obtained and sequenced. This fragment contained an open reading frame (ORF) whose amino acid sequence was highly similar to the sequences of other known PHB synthase genes, especially to a synthase from Azospirillum brasilense (74% identity).