The F420S substitution enhances the specific activity of Ralstonia eutropha PHA synthase (PhaC(Re)). We have now carried out site-directed saturation mutagenesis of F420 of PhaC(Re) and, amongst the F420 mutants, the F420S mutant gave the highest poly(3-hydroxybutyrate) (PHB) content. In vitro activity assay showed that the F420S enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. Enhancement of PHB accumulation was achieved by combination of the F420S mutation with a G4D mutation, which conferred high PHB content and high in vivo concentration of PhaC(Re) enzyme. The G4D/F420S mutant gave a higher PHB content and in vivo concentration of PhaC(Re) enzyme than the F420S mutant, while the molecular weight of the PHB polymer of the double mutant was similar to that of the F420S mutant.