Exploring phasin-polyhydroxyalkanoate interactions through in vivo and in vitro binding assays
Year: 2026
Type: article
Abstract: 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. Distinct functional roles of phasin families in PHA granule assembly. The study reveals that granule architecture is dictated by the specific phasin-polymer interplay. PhaP1 (green) functions as a high-affinity surfactant that binds scl-PHA immediately, promoting the formation of single granule. In contrast, PhaI (purple) displays a cooperative binding threshold. In vivo , PhaI acts as an active segregation factor, driving the formation of multiple, size-homogeneous granules. These findings establish PhaP1 as a superior tag for high-density ligand display and PhaI as a tool for engineering nanoparticle population homogeneity. • Phasins modulate PHA granule number and morphology in vivo . • PhaP1 and PhaI show selective binding to different PHA types. • Phasins enable functionalization of PHA for biotech applications.
Language: English
Subfield: Biomaterials
Field: Materials Science
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $4,390
OpenAlex ID: W7124446493