MOLECULAR ANALYSIS OF THE POLY(3-HYDROXYALKANOATE) SYNTHASE GENE FROMA METHYLOTROPHIC BACTERIUM, PARACOCCUS-DENITRIFICANS

A 3.6-kb EcoRI-SnlI fragment of Paracoccus denitrificans DNA hybridize d with a DNA probe carrying the poly(3-hydroxyalkanoate) (PHA) synthas e gene (phaC) of Alcaligenes eutrophus. Nucleotide sequence analysis o f this region showed the presence of a 1,872-bp open reading frame (OR F), which corresponded to a polypeptide with a molecular weight of 69, 537. Upstream of the ORF, a promoter-like sequence was found. Escheric hia coli carrying the fusion gene between lacZ and the ORF accumulated a level of poly(3-hydroxybutyrate) that was as much as 20 wt% of the cell dry weight in the presence of beta-ketothiolase and acetoacetyl-c oenzyme A reductase genes of A. eutrophus. The ORF was designated phaC (Pd). A plasmid vector carrying the phaC(Pd)-lacZ fusion gene downstre am of the promoter-like sequence expressed P-galactosidase activity in P. denitrificans, When a multicopy and broad-host-range vector carryi ng the ORF along with the promoter-like sequence was introduced into P . denitrificans, the PHA content in the cells increased by twofold com pared with cells carrying only a vector sequence.