Phasins PhbP2 and PhbP3 Are Involved in the Depolymerization of Polyhydroxybutyrate in Azotobacter vinelandii

Polyhydroxybutyrate (PHB) is a natural polyester synthesized and stored intracellularly as cytoplasmic granules, serving as a carbon and energy reserve in certain bacteria. These granules are surrounded by a variety of proteins, including those directly involved in PHB metabolism-both in its synthesis and degradation-as well as a group of non-enzymatic proteins known as phasins, which constitute the major protein component of the PHB granule. Although phasins lack enzymatic activity, some, such as PhbP1 in Azotobacter vinelandii, significantly influence granule size and number, while others have been shown to modulate enzymes involved in PHB synthesis or degradation in PHB-producing bacteria. This study aimed to elucidate the roles of phasins PhbP2 and PhbP3 in PHB metabolism in A. vinelandii. Our findings indicate that PhbP3 belongs to a novel family of phasin proteins and demonstrate that, although PhbP2 and PhbP3 do not contribute to granule structure, they are involved in PHB degradation. Gene inactivation of phbP2 and phbP3 led to a decrease in PHB depolymerase activity. Additionally, SDS-PAGE analysis revealed alterations in the relative abundance of some granule-associated proteins in the mutants, suggesting that PhbP2 and PhbP3 may play a role in stabilizing proteins on the PHB granule surface.