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

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) 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. However, an in vitro assay revealed that the surface of GST-fused PhaC (GST-PhaC) had increased hydrophilicity, and tended to form correct PhaC dimers when added to the (R)-3-hydroxybutyryl-CoA substrate. Although GST-PhaC displayed a long lag phase at the start of a polymerization reaction, granule-associated GST-PhaC showed higher catalytic activity than PhaC in kinetic analysis. The results are discussed in light of the dimerization mechanisms of PhaC.