Exploiting Phase Partitioning of a Giant Trimeric Fibrous Protein Scaffold to Potentiate Chitinase‐Driven Antifungal Activity

Recently, the relationship between phase-separation behavior and biological functions in both natural and artificial systems has garnered significant attention. An aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and dextran (Dex), traditionally utilized for protein purification, has emerged as a model for liquid-liquid phase separation (LLPS). In this study, we found that Cstalk, a giant trimeric fibrous protein with a length of approximately 40 nm, preferentially partitions to the Dex-rich phase in PEG/Dex ATPS. Consequently, conjugation of a protein of interest (POI) to the giant protein scaffold by the SpyTag-SpyCatcher system enabled the recruitment of POIs into the Dex-rich phase. Given that the Dex phase acts as a mimic of a polysaccharide-rich fungal cell wall, we exploited the potential of Cstalk to deliver antifungal chitinase to the cell wall of Trichoderma viride. Precise control of the conjugation ratio of chitinase to Cstalk showed the tunable antifungal activity against the growth of T. viride depending on the enzyme loading. Amidst the urgent need for novel antifungal strategies, our results demonstrated a new design principle of antifungal therapeutics by leveraging the unique phase-selective partitioning property and the tunable protein loading capacity of a giant fibrous protein scaffold.