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Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer {poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]} consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In thephb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), β-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed byphbB Ps-phbA Ps-phbC Ps. Upstream from phbB Ps was found thephbR Ps gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbR Ps gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbR Ps in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of β-galactosidase activity from a transcriptional phbpromoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPs is a positive regulatory protein controlling the transcription of phbBAC Ps in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) were flanked by a PHA depolymerase gene (phaZ Ps), and two adjacent open reading frames (ORF1 and phaD Ps), and the gene order was ORF1, phaC1 Ps,phaZ Ps, phaC2 Ps, andphaD Ps. Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida andRalstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.

Pseudomonassp. 61-3 accumulated poly(3-hydroxyalkanoates), P(3HA), in nitrogen-free media containing medium- and long-chain alkanoic acids or plant oils. The polyesters of 3HA units of even carbon numbers ranging from C4 to C12 were produced from the alkanoic acids of even carbon numbers, while those produced from the alkanoic acids of odd carbon numbers contained 3-hydroxybutyrate (3HB) and 3HA units of odd carbon numbers of C5 to C11. The strain produced simultaneously P(3HB) homopolymer and a random copolymer of 3HA units of C4 to C12 within cells from the alkanoic acids of even carbon numbers, while a copolymer of 3HB and 3-hydroxyvalerate was formed with a random copolymer of 3HA units from the alkanoic acids of odd carbon numbers. The results suggest that Pseudomonas sp. 61-3 has two types of PHA synthases with different substrate specificities.

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Pseudomonas sp. 61‐3 is able to produce a blend of poly(3‐hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer [P(3HB‐co‐3HA)] consisting of 3‐hydroxyalkanoate units of 4–12 carbon atoms. In a cell accumulating polyhydroxyalkanoates upon glucose or alkanoic acids, both needle‐type and mushroom‐type structures were observed as PHA granules by freeze‐fracture electron microscopy, indicating that Pseudomonas sp. 61‐3 synthesized and stored both P(3HB) and P(3HB‐co‐3HA) granules simultaneously as separate granules in the same cell. 13C‐NMR analysis of polyhydroxyalkanoates synthesized from 13C‐labeled octanoate revealed that 3‐hydroxybutyrate units in the resultant polyhydroxyalkanoates were not only supplied via fatty acid β‐oxidation but also via dimerization of two acetyl‐CoA molecules in Pseudomonas sp. 61‐3. Approximately 26% of 3‐hydroxybutyrate units was found to be generated via dimerization of acetyl‐CoA when octanoate was fed as a carbon source.