In vivo characterization of FadB homologous enzymes including PaaG, YdbU and YgfG for medium-chain-length(MCL) Polyhydroxyalkanoates(PHA) biosynthesis was carried out in fadB mutant Escherichia coli. Previously, it was reported that amplificaion of FadB homologous enzyme such as PaaG and YdbU in fadB mutnat E.coli resulted in enhanced biosynthesis of MCL-PHA by greater than two fold compared with control strain. In this study, we constructed paaG fadB double mutant E.coli WB114 and ydbU fadB double mutant E.coli WB115 to investigate the roles of PaaG and YdbU in biosynthesis of MCL-PHA. Inactivation of paaG and ydbU genes in fadB mutant E.coli harboring Pseudomonas sp.61-3 phaC2 gene reduced the MCL-PHA production to 0.16 and 0.16 PHA g/L, respectively from 2 g/L of sodium decanoate, which are much lower than 0.43 PHA g/L obtained with fadB mutant E.coli WB101 harboring the phaC2 gene. Also, we identified new FadB homologous enzyme YgfG, and examined its roles by overexpression of ygfG and construction of ygfG fadB double mutant E.coli WB113.