Genetic Analysis of Comamonas acidovorans Polyhydroxyalkanoate Synthase and Factors Affecting the Incorporation of 4-Hydroxybutyrate Monomer

The polyhydroxyalkanoate (PHA) synthase gene ofComamonas acidovorans DS-17 (phaCCa ) was cloned by using the synthase gene ofAlcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbpSma I-Hin dIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp β-ketothiolase gene (phaACa ). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes inA. eutrophus . The arrangement of PHA biosynthesis genes inC. acidovorans was also similar to that inA. eutrophus except that the third gene,phaB , coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream ofphaACa . The cloned fragment complemented a PHA-negative mutant ofA. eutrophus , PHB− 4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase ofC. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that inC. acidovorans , it is the metabolic pathway, and not the specificity of the organism’s PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.