Two PHA synthase phaC1 and phaC2 genes cloned from the new strain Pseudomonas putida KCTC1639 were metabolically engineered for the overproduction of medium-chain-length polyhydroxyalkanoate (mcl-PHA). The overexpressed phaC1 and phaC2 genes in P. putida KCTC1639 were compared in terms of the biosynthesis of mcl-PHA, fatty acid assimilation, distribution of 3-hydroxylacyl monomer units, granular morphology, and thermophysical properties of the accumulated mcl-PHA. The biosynthesis of mcl-PHA was enhanced only by the overexpressed phaC1 gene up to 2.86-fold, in contrast, the phaC2 gene did not activate the biosynthesis of mcl- PHA. The overexpressed phaC1 gene tended to form enlarged, high molecular weight, and lower crystalline mcl-PHA granules, whereas the amplified phaC2 gene induced the fragmentation of mcl-PHA into a few small-sized granules. The transformant P. putida KCTC1639 overexpressing the phaC1 gene encoding PHA synthase I was cultivated by pH-stat fed-batch cultivation, and the concentration and content of mcl-PHA increased up to 8.91 g L-1 and 70.5%, respectively.