To efficiently produce polyhydroxyalkanoates (PHAs) from substrate mixture containing various carbon sources, Escherichia coli phosphotransferase system (PTS) mutants were constructed, in which the carbon catabolite repression in the presence of glucose was released. When cultivated in the medium containing glucose and xylose, E. coli LR1010, harboring phaC(Re) and phaAB(Re) genes from Ralstronia eutropha, could simultaneously consume glucose and xylose and accumulate short-chain-length PHA, whereas E. coli LR1120 and LR1110, harboring phaC1 gene from Pseudomonas aeruginosa, could accumulate medium-chain-length PHA. At the same time, LR1110 could simultaneously utilize the mixture of glucose and fatty acids. Semiquantitative reverse transcription polymerase chain reaction analysis indicated that disrupting phosphoenolpyruvate: sugar PTS in this strain released the repression on fad genes.