Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate (HD), and 3-hydroxydodecanoate (HDD) from a wide-range of carbon sources. In this study, fadA and fadB genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KT2442 were knocked out to weaken the beta-oxidation pathway. Two-step culture was proven as the optimal method for PHA production in the mutant termed P. putida KTOY06. In a shake-flask culture, when dodecanoate was used as a carbon source, P. putida KTOY06 accumulated 84 wt % PHA, much higher than 50 wt % PHA in its wild type KT2442. The PHA monomer composition was completely different: the HDD fraction in PHA produced by KTOY06 was 41 mol %, much higher compared with 7.5 mol % only in KT2442. The fermentor-scale culture indicated the HDD fraction in PHA decreased during the culture time from 35 to 25 mol % in a one-step fermentation process or from 75 to 49 mol % in a two-step fermentation process. It is for the first time that PHA with a dominant HDD fraction was produced. Thermal and mechanical properties assays indicated that this new type PHA with a high HDD fraction had higher crystallinity and tensile strength than PHA with a low HDD fraction did, demonstrating an improved application property.