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

## TL;DR

This study shows that AA10 LPMO domains from different bacteria consistently form inclusion bodies in E. coli, suggesting they can be used to design stable protein nanoparticles.

## Contribution

The aggregation tendency of AA10 LPMO domains is shown to be conserved across multiple species, expanding their potential for biotechnological applications.

## Key findings

- Four AA10 homologues from different species formed uniform inclusion bodies in E. coli.
- The inclusion bodies had sub-micron diameters and β-sheet-rich structures.
- Most of the expressed protein partitioned into the insoluble fraction, indicating strong aggregation.

## Abstract

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 (Ccelp40) consistently forms IBs and, when fused to diverse proteins, generates functional IBs. Here, we examined whether this strong aggregation propensity is unique to Ccelp40 or a broader feature of AA10 LPMOs. Four homologous domains from phylogenetically distinct microorganisms, Kallotenue papyrolyticum (Kpapp40), Kibdelosporangium aridum (Karip40), Archangium lipolyticum (Alipp40), and Phytohabitans suffuscus (Psufp40), 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 Ccelp40 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.