Bifunctional Polyester Synthase–Channel Driving Phosphorylated PHB–PHV Synthesis and Ion Conductance

Microbial polyhydroxyalkanoates (PHAs) have traditionally been viewed as inert carbon reserves, yet emerging evidence implicates these polyesters in ion transport and stress response. Here, we identify and characterize ORF1 from Hanseniaspora valbyensis as the first genetically encoded polyester synthase–translocator that couples phosphorylated poly[(R)-3-hydroxybutyrate–3-hydroxyvalerate] (PHB–PHV) biosynthesis with vectorial export through a membrane pore. The 344residue ORF1 protein was heterologously expressed in Escherichia coli ΔphaC, and its product validated by TEM, FTIR, NMR and GC–MS. Six independent topology predictors (Phobius, PolyPhobius, MEMSATSVM, Philius, DeepTMHMM and MemBrain) and a TOPCONS2 metaanalysis converged on a central multihelix region (residues 159–222) as the membraneembedded core. AlphaFold2 modeling and PoreWalker analysis revealed an amphipathic channel with alternating hydrophobic clamps and electrostatic constrictions (SDUS geometry). PrankWeb pocket mapping and SwissDock simulations demonstrated that divalent cations (Ca²⁺, Mg²⁺), ATP and phosphorylated PHB–PHV oligomers occupy overlapping binding corridors, stabilized by aromatic, aliphatic and basic residues. Spectroscopic signatures of phosphate incorporation and aliphatic backbone structure corroborated in silico interaction models. This bifunctional architecture elevates PHAs from metabolic stores to active mediators of membrane homeostasis and stress adaptation. ORF1 defines a new class of polyesterbased channels, unifying biopolymer synthesis and transport within a single molecular scaffold, and offers a platform for engineering bespoke polymer conduits in synthetic biology.