phaJ and fabG genes encoding (R)-specific 2-enoyl-CoA hydratase and 3-ketoacyl-acyl carrier protein reductase, respectively, were cloned from Pseudomonas putida KCTC1639 and amplified in the parent strain to achieve oversupplementation of (R)-3-hydroxyalkanoate monomer units for the biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA). The comparative effect of the overexpressed fahG and phaJ genes in P pulida KCTC1639 on the biosynthesis of mcl-PHA and the cell growth were elucidated. Overexpression of phaJ enhanced the biosynthesis of mcl-PHA, increasing its content and concentration from 18% to 27% and 0.38 to 0.51 g/l, respectively. Conversely, fabG overexpression tended to depress the biosynthesis of mcl-PHA, possibly due to the reversible conversion of (R)-3-hydroxyalkanoate monomer units into 3-ketoacyl-CoA.