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