Cloning of poly(3-hydroxybutyric acid) synthase genes ofRhodobacter sphaeroidesandRhodospirillum rubrumand heterologous expression inAlcaligenes eutrophus

From genomic libraries of the purple non-sulfur bacteria Rhodospirillum rubrum Ha and Rhodobacter sphaeroides ATCC 17023 in the broad-host range cosmid pVK100, we cloned a 15- and a 14-kbp Hin dIII restriction fragment, respectively. Each of these fragments restored the ability to accumulate poly(3-hydroxybutyrate) (PHB), in the PHB-negative mutant Alcaligenes eutrophus PHB−4. These hybrid cosmids also complemented PHB-negative mutants derived from wild-type R. rubrum or R. sphaeroides. Both fragments hybridized with the PHB synthase structural gene of A. eutrophus H16 and conferred the ability to express PHB synthase activity. Only the 15-kbp Hin dIII fragment from R. rubrum conferred on the mutant PHB−4 the ability to form large PHB granules (length up to 3.5 μm).