Metabolic Engineering of a Novel Propionate-Independent Pathway for the Production of Poly(3-Hydroxybutyrate- <i>co</i> -3-Hydroxyvalerate) in Recombinant <i>Salmonella enterica</i> Serovar Typhimurium

ABSTRACT A pathway was metabolically engineered to produce poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBV), a biodegradable thermoplastic with proven commercial applications, from a single, unrelated carbon source. An expression system was developed in which a prpC strain of Salmonella enterica serovar Typhimurium, with a mutation in the ability to metabolize propionyl coenzyme A (propionyl-CoA), served as the host for a plasmid harboring the Acinetobacter polyhydroxyalkanoate synthesis operon ( phaBCA ) and a second plasmid with the Escherichia coli sbm and ygfG genes under an independent promoter. The sbm and ygfG genes encode a novel (2 R )-methylmalonyl-CoA mutase and a (2 R )-methylmalonyl-CoA decarboxylase, respectively, which convert succinyl-CoA, derived from the tricarboxylic acid cycle, to propionyl-CoA, an essential precursor of 3-hydroxyvalerate (HV). The S. enterica system accumulated PHBV with significant HV incorporation when the organism was grown aerobically with glycerol as the sole carbon source. It was possible to vary the average HV fraction in the copolymer by adjusting the arabinose or cyanocobalamin (precursor of coenzyme B 12 ) concentration in the medium.