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Poly[beta-hydroxybutyrate-co-beta-hydroxyvalerate] co-polymer, PHBV, is a polyhydroxyalkanoate (PHA) that has greater utility as a biodegradable thermoplastic polyester than poly-beta-hydroxybutyrate, PHB. In order to produce PHBV, a system of pathways is required to produce both hydroxybutyrate (HB) and hydroxyvalerate (HV) monomers from the sources of carbon. A working model for conversion of glucose to PHBV via acetyl- and propionyl-coenzyme A was constructed by expressing the PHA biosynthesis genes from Alcaligenes eutrophus in Escherichia coli strain K-12 under novel growth conditions. When 1 mM valine was added to 1% glucose medium, growth ceased and up to 2.5% of the incorporated monomers were HV; up to 4% were HV when 1 mM threonine was added as well. Threonine dehydratase (TD) converts threonine to alpha-ketobutyrate; TD is required for HV to be incorporated into PHA unless its transaminated reaction product, alpha-aminobutyrate, is added to the medium. Intracellular alpha-ketobutyrate accumulates when valine is added to the medium because valine, which cannot be metabolized to HV by E. coli strain K-12, stimulates TD and inhibits acetolactate synthase. In turn, alpha-ketobutyrate is converted to propionyl-CoA by the E. coli pyruvate dehydrogenase complex. This constitutes a defined system of pathways for synthesis of a heteropolymeric PHA from a single carbon source, which in the future could be transferred to other organisms including plants.