Gelphyman, a cryptic and novel thermoreversible gelling polysaccharide activated by c-di-GMP in Paraburkholderia phymatum STM815.

Cyclic di-GMP is a key bacterial second messenger that regulates polysaccharide biosynthesis and biofilm formation. Here, we report the discovery and characterization of Gelphyman, a novel thermoreversible gelling polysaccharide produced by Paraburkholderia phymatum STM815 under artificially elevated c-di-GMP levels. Heterologous expression of the diguanylate cyclase PleD* triggered a c-di-GMP-dependent aggregative phenotype and induced production of a cryptic, cell envelope-associated polymer that could be purified upon heat treatment. Gelphyman forms hydrogels at concentrations as low as 0.3 mg/mL through a temperature-driven, ion-independent mechanism. Structural analyses (GC-MS, NMR) revealed a unique branched tetrasaccharide repeating unit composed of →3)-α-d-Manp-(1 → 3)-β-d-Glcp-(1 → 2)-α-d-Rhap-(1→, with an α-6-deoxy-l-talopyranose side branch linked to Rhap at C-3. Differential scanning calorimetry confirmed thermoreversible gelation (gelation at ∼36 °C; gel-sol transition at ∼65 °C). Rheology indicated elastic and shear-thinning hydrogels with network efficiency comparable to agarose. Notably, the lowest tested concentration is one order of magnitude below the theoretical overlap concentration for coil state at high temperature, suggesting that a thermally induced conformational expansion on cooling drives network formation. Morphological studies revealed a fibrillar 3D network with concentration-dependent pore size. This work positions Gelphyman as a promising biopolymer with potential long-term applications in sustainable biotechnology, food formulations, and advanced biomaterials.