Four (R)-specific enoyl CoA hydratases (PhaJ) interconnect the beta-oxidation pathway with PHA biosynthesis in Pseudomonas aeruginosa. The use of antisense technique and over-expression to delineate the role of two of these enzymes, PhaJ1 and PhaJ4 forms the basis of this study. It has been observed that P. aeruginosa recombinant with phaJ1 antisense construct, fed with different fatty acids, produces PHA with less hydroxy octanoate (7-11% reduction) and a proportionate increase in other monomer fractions, compared to that of the control. Recombinants bearing phaJ4 antisense construct are found to contain less hydroxy decanoate (10-11% reduction) and more or less equal amount of hydroxy octanoate, compared to that of the control. P. aeruginosa has produced PHA with more hydroxy octanoate and decanoate (6-17% increase), respectively, when PhaJ1 and PhaJ4 have been over-expressed individually or along with PhaC1. PhaJ1 and PhaJ4 are found to be involved mainly in the production of hydroxy octanoyl CoA and hydroxy decanoyl CoA, respectively, in P. aeruginosa. The strongest accumulation of hydroxy octanoate and hydroxy decanoate has been observed along with hydroxy butyrate, in PHA, produced by E. coli, when PhaC1 has been co-expressed with PhaJ1 and PhaJ4, respectively. We have demonstrated, for the first time, the polymerization of hydroxy butyryl CoA monomers in recombinant E. coli by PhaC1 of P. aeruginosa.