Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli was expressed together with polyhydroxyalkanoate synthase gene (phaC(Ac)) and (R)-enoyl-CoA hydratase gene (phaJ(Ac)) from Aeromonas caviae. The expression plasmids were introduced into E. coli JM109, DH5 alpha and XL1-blue, respectively. Compared with the strains harboring only phaC(Ac) and phaJ(Ac), all recombinant E. coli strains harboring yafH, phaC(Ac) and phaJ(Ac) accumulated at least four times more poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Cell dry weights produced by all recombinants containing yafH were also considerably higher than that without yafH. The addition of acrylic acid which serves as inhibitor for beta-oxidation and may lead to more precursor supply for PHA synthesis did not result in improved PHBHHx production compared with that of the overexpression of yafH. It appeared that the overexpression of acyl-CoA dehydrogenase gene (yafH) enhanced the supply of enoyl-CoA which is the substrate of (R)-enoyl-CoA hydratase. With the enhanced precursor supply, the recombinants accumulated more PHBHHx.