DISCOVERY AND BIOCHEMICAL CHARACTERIZATION OF A FUNGAL ICE NUCLEATION PROTEIN FROM PODILA CLONOCYSTIS

Biological ice nucleation plays a pivotal role in atmospheric processes, yet the molecular basis of fungal ice nucleation remains poorly understood compared to bacterial systems. Here, we report the biochemical characterization of an ice nucleation protein (PcINP) from a soil-dwelling fungus Podila clonocystis, not previously reported to produce ice nuclei. Using sequence similarity network analysis, we identified PcINP as a putative fungal homolog of bacterial ice nucleation proteins and confirmed its function through recombinant expression in Escherichia coli. We probe the function of PcINP structure through domain truncations and demonstrate that a poorly structured N-terminal region is not necessary for ice nucleation activity and can be functionally replaced with an expression enhancing SUMO fusion tag. Finally, we observe both monomeric and aggregated PcINP in E. coli lysates using SEC-MALS but are unable to distinguish their ice nucleation activity pointing to an unknown in vitro aggregation mechanism. Our findings establish PcINP within the emerging class fungal ice nucleation protein with distinct structural features and high stability, expanding the known diversity of biological ice nucleators and highlighting their potential for environmental and biotechnological applications.