Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems

A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATC C 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower h omology to the R. rubrum strain Ha PHA synthase than would be expected with in the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutr ophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-syntha se-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 m onomers in the polymer when fermented in medium supplemented with 0.1% hept anoate as the sole carbon source. When the T. vi olacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PH A synthase of R. eutropha, when the transconjugants were fermented in mediu m supplemented with 0.4% propionate. Evaluation for preference of medium-ch ain-length monomers demonstrated the flexibility of the N. corrallina, T. v iolacea, and R. eutropha synthase genes to polymerize a copolyester compose d of short- and medium-chain-length monomers when the respective transconju gants were fermented in medium supplemented with 0.5% octanoate. These stud ies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and med ium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observa tions suggest that the composition of the PHA from the PHA-producing organi sms does not necessarily reflect the inherent specificity of the PHA syntha se.