PHA SYNTHASE ACTIVITY CONTROLS THE MOLECULAR-WEIGHT AND POLYDISPERSITY OF POLYHYDROXYBUTYRATE IN-VIVO

A synthetic operon for polyhydroxyalkanoate (PHA) biosynthesis designe d to yield high levels of PHA synthase activity in vivo was constructe d by positioning a genetic fragment encoding beta-ketothiolase and ace toacetyl-CoA reductase behind a modified synthase gene containing an E scherichia coli promoter and ribosome binding site. Plasmids containin g the synthetic operon and the native Alcaligenes eutrophus PHA operon were transformed into E. coli DH5 alpha and analyzed for polyhydroxyb utyrate production. The molecular weight of polymer isolated from reco mbinant E. coli containing the modified synthase construct; determined by multiangle light scattering, was lower than that of the polymer fr om E. coli containing the native A. eutrophus operon. A further decrea se in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon. Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain enc oding the native operon with a strain possessing the synthetic operon indicates that the amount of polyhydroxyalkanoate synthase in a host o rganism plays a key role in controlling the molecular weight and the p olydispersity of polymer.