Sequence-Encoded Aggregation of AA10 LPMO Domains as a Basis for Inclusion Body Design

Inclusion bodies (IBs) in Escherichia coli are increasingly recognised as nanostructured materials with tunable morphology and functional potential. The N-terminal auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenase (LPMO) domain from Caldibacillus cellulovorans (Ccel_p40) consistently forms IBs and, when fused to diverse proteins, generates functional IBs. Here, we examined whether this strong aggregation propensity is unique to Ccel_p40 or a broader feature of AA10 LPMOs. Four homologous domains from phylogenetically distinct microorganisms, Kallotenue papyrolyticum (Kpap_p40), Kibdelosporangium aridum (Kari_p40), Archangium lipolyticum (Alip_p40), and Phytohabitans suffuscus (Psuf_p40), were heterologously expressed in E. coli under identical cytosolic conditions. All homologues accumulated predominantly in the insoluble fraction, forming morphologically uniform IBs with sub-micron diameters (550-860 nm) and moderate polydispersity indices (0.45-0.54). SDS-PAGE densitometry indicated that most of each expressed protein partitioned into the insoluble fraction. Field-emission scanning electron microscopy revealed compact spherical aggregates, and Fourier-transform infrared spectroscopy showed β-sheet-enriched secondary structures characteristic of ordered IBs. These results indicate that the pronounced aggregation tendency previously observed for Ccel_p40 is conserved across the AA10 homologues examined. The findings support the view that the AA10 domain represents a promising scaffold for generating stable, recyclable protein nanoparticles and provides a comparative basis for future IB-based biotechnological designs.