Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters synthesized by various bacteria under nutrient-limiting conditions with excess carbon. These polymers accumulate as intracellular granules coated with granule-associated proteins (GAPs), including phasins, the most abundant and functionally diverse GAPs. Phasins play essential roles in granule formation, PHA metabolism, and have emerged as promising tools for functionalizing PHA surfaces in applications such as enzyme immobilization and drug delivery. In this study, we assessed the binding affinity and influence of two phasins, PhaP1 and PhaI, on PHA accumulation and granule morphology in three engineered Pseudomonas putida KT2440 strains (PP00_01, PP01_02, and PP05_17), optimized for tailored PHA production with distinct monomer compositions (medium-, short-chain-length PHAs, and the hybrid scl/mcl-PHA producer, respectively). Phasin-msfGFP fusion proteins were expressed and monitored using in vivo fluorescence binding assays. PHA accumulation was evaluated via microscopy and GC-MS. Despite similar PHA yields (%PHA/CDW) in strains with and without phasins, PhaI expression in mcl-PHA-producing strain PP00_01 led to increased granule number and homogeneity, confirmed by transmission electron microscopy. Additionally, flow cytometry analyses revealed differential surface affinities of phasins depending on polymer type, indicating that both phasin type and PHA physicochemical properties influence binding strength. PhaP1 and PhaI displayed distinct preferences for scl- and mcl PHAs, respectively. Overall, this work underscores the dual role of phasins in modulating PHA production and enabling selective functionalization of PHA granules, supporting their use as bio-affinity tags for polymer-based biotechnological applications.