A bacterial strain capable of synthesizing polyhydroxyalkanoates (PHAs) with an unusual pattern of monomer units was isolated from activated sludge using the enrichment culture technique. The organism, identified as Pseudomonas aeruginosa P-5, produced polyesters consisting of 3-hydroxyvalerate and medium-chain-length (MCL) 3-hydroxyalkanoate monomer units when C-odd alkanoic acids such as nonanoic acid and heptanoic acid were fed as the sole carbon source. Solvent fractionation experiments using chloroform and hexane revealed that the 3-hydroxyalkanoate monomer units in these polyesters were copolymerized. The molar concentration of 3-hydroxyvalerate in the polyesters produced were significantly elevated up to 26 mol% by adding 1.0 g/L valeric acid as the cosubstrate. These copolyesters were sticky with low degrees of crystallinity. The PHA synthase genes were cloned, and the deduced amino acid sequences were determined. P. aeruginosa P-5 possessed genes encoding MCL-PHA synthases (PhaC1 and PhaC2) but lacked the short-chain-length PHA synthase gene, suggesting that the MCL-PHA synthases from P. aeruginosa P-5 are uniquely active for polymerizing (R)-3-hydroxyvaleryl-CoA as well as MCL (R)-3-hydroxyacyl-CoAs.

Pseudomonas aeruginosa P-5 is an unusual organism capable of synthesizing polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyvalerate (3HV) and medium-chain-length (MCL) 3-hydroxyalkanoate (3HA) monomer units when C-odd alkanoic acids are fed as the sole carbon source. Evaluation of the substrate chain-length specificity of two P. aeruginosa P-5 PHA synthases (PhaC1P-5 and PhaC2P-5) by heterologous expression of PhaC1P-5 and PhaC2P-5 genes in Pseudomonas putida GPp104 revealed that PhaC2P-5 incorporates both 3HV and MCL 3HAs into PHA, whereas PhaC1P-5 favors only MCL 3HAs for polymerization. In order to obtain PhaC2P-5 mutants with altered substrate specificity, site-specific mutagenesis for PhaC2P-5 was conducted. Amino acid substitutions of PhaC2P-5 at two positions (Ser326Thr and Gln482Lys) were very effective for synthesizing copolymers with a higher 3HV fraction. When recombinant P. putida GPp104 harboring double mutated phaC2P-5 gene (phaC2P-5QKST ) was grown on nonanoic acid, 2.5-fold increase of copolymer content with 3.8-fold increase of 3HV fraction was observed. The phaC2P-5QKST-containing Ralstonia eutropha PHB-4 supplemented with valeric acid also produced copolymers consisting of 3HV and 3-hydroxyheptanoate with a high 3HV fraction. These results suggest that recombinants containing phaC2P-5QKST could be useful for production of new PHA copolymers with improved material properties.Key words: Pseudomonas aeruginosa P-5, polyhydroxyalkanoate synthases, site-directed mutagenesis, substrate specificity