Genetic Characterization of the Poly(hydroxyalkanoate) Synthases of Various Pseudomonas oleovorans Strains

We identified the poly(hydroxyalkanoate) synthase (PHAS) genes of three strains of Pseudomonas oleovorans by using polymerase chain reaction (PCR)-based detection methods. P. oleovorans NRRL B-14682 contains Class I PHA synthase gene (phaC), NRRL B-14683 harbors Class II phaC1 and phaC2 genes, and NRRL B-778 contain both the Class I and II PHA synthase genes. Inverse-PCR and chromosomal walking techniques were employed to obtain the complete sequences of the Class I phaCs of NRRL B-778 (phbC₇₇₈; 1698 bps) and B-14682 (phbC₁₄₆₈₂; 1899 bps). BLAST search indicated that these genes are new and had not been previously cloned. The gene product of phbC₇₇₈ (i.e., PhbC₇₇₈; 566 amino acid residues) is homologous to the Class I PHA synthases of Pseudomonas sp. HJ-2 and Pseudomonas sp. strain 61-3, and that of phbC₁₄₆₈₂ (PhbC₁₄₆₈₂; 632 amino acids) is homologous to PHAS of Delftia acidovorans. The PhbC₁₄₆₈₂ contains an extra sequence of 33 amino acids in its conserved α/β-hydrolase domain, making it only the second Class I PHA synthase found to contain this cellular proteolytic sequence. Consistent with their Pseudomonas origin, the codon-usage profiles of PhbC₇₇₈ and PhbC₁₄₆₈₂ are similar to those of Pseudomonas Class II PHASs. These new Pseudomonas Class I phbC genes provide valuable addition to the gene pool for the construction of novel PHASs through gene shuffling.