Biosynthesis pathway of medium-chain-length(MCL) polyhydroxyalkanoates (PHA) from fatty acid β-oxidationpathway was constructed in recombinant Escherichiacoli by introducing the Pseudomonas sp. 61-3 PHA synthasegene (phaC2) and the maoC genes from Pseudomonas putida,Sinorhizobium meliloti, and Ralstonia eutropha. The metaboliclink between fatty acid β-oxidation pathway and PHAbiosynthesis pathway was constructed by MaoC, which ishomologous to P. aeruginosa (R)-specific enoyl-CoA hydratase(PhaJ1). When the E. coli W3110 strains expressing thephaC2 gene and one of the maoC genes from P. putida, Sinorhizobiummeliloti, and Ralstonia eutropha were cultured inLB medium containing 2 g/L of sodium decanoate as a carbonsource, MCL-PHA that mainly consists of 3-hydroxyhexanoate(3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate(3HD), was produced. The monomer composition ofPHA and PHA contents varied depending on MaoC employedfor the production of PHA. The highest PHA content of 18.7wt% was achieved in recombinant E. coli W3110 expressingthe phaC2 gene and the P. putida maoC gene. These resultssuggest that MCL-PHA biosynthesis pathway can be constructedin recombinant E. coli strains from the b-oxidation pathwayby employing MaoC able to supply (R)-3-hydroxyacyl-CoA, the substrate of PHA synthase.