In vivo and in vitro characterization of hydrophilic protein tag-fused Ralstonia eutropha polyhydroxyalkanoate synthase

Abstract Polyhydroxyalkanoates (PHAs) are synthesized by bacteria as an intracellular storage polyester, where PHA synthase (PhaC) catalyzes the polymerization of its substrate hydroxyacyl-coenzyme A (HA-CoA) to form PHA. When PhaC is overexpressed in Escherichia coli , most PhaC protein is produced as insoluble inclusion bodies due to its low aqueous solubility. This study aimed to improve the solubility of Ralstonia eutropha PHA synthase (PhaC Re ) by fusing a hydrophilic tag, glutathione S -transferase (GST), to the protein's N-terminus. In in vivo assays, the GST tag had no obvious effect on solubility and enzymatic activity of PhaC Re . However, an in vitro assay revealed that the surface of GST-fused PhaC Re (GST-PhaC Re ) had increased hydrophilicity, and tended to form correct PhaC Re dimers when added to the ( R )-3-hydroxybutyryl-CoA substrate. Although GST-PhaC Re displayed a long lag phase at the start of a polymerization reaction, granule-associated GST-PhaC Re showed higher catalytic turnover than PhaC Re in kinetic analysis. The results are discussed in light of the dimerization mechanisms of PhaC Re .