Functional and Structural Characterisation of Expansins from Neocallimastigomycetes

Developing sustainable strategies for lignocellulose utilisation is vital for reducing environmental impact and advancing biobased products. This thesis investigates microbial expansin- and loosenin-like proteins (EXLXs and LOOLs) as potential contributors to improved plant biomass deconstruction. The study aims to functionally and structurally characterise EXLXs and LOOLs from Neocallimastigomycota, providing insight into their roles in lignocellulose modification and their potential to enhance biomass processing.

Four EXLXs and four LOOLs from Neocallimastix californiae, N. lanati and Piromyces finnis are selected to represent both organism and sequence variation, and are heterologously expressed in Pichia pastoris. Their cellulose binding, synergy with cellulases, and potential hidden enzymatic activity requiring an exogenous nucleophile are examined. Structural analyses, including circular dichroism, nanoDSF, NMR spectroscopy, and X-ray crystallography, reveal domainlevel organisation and ligandspecific interactions.

In cellulase synergy assays, NlEXLX1 and NlLOOL1 enhance glucose release from Avicel, with NlLOOL1 most effective at 5–50 μM and NlEXLX1 at 0.5 μM. Enhancement is also observed with BSA, but experimental evidence suggests that expansin-mediated enhancement arises through substrate modification, whereas BSA acts by stabilising cellulase activity. The exogenous nucleophile hypothesis show inconclusive results. Structural studies show that NlLOOL1 possesses flexible loops and a βbarrel core consistent with AlphaFold predictions, with the ligninmodel compound guaiacylglycerolβguaiacyl ether (GBG) interacting at a conserved site comprising residues A72, K110, C111, V113, C114, and D120. The crystal structure of NlEXLX1 reveals two xylosebinding sites, one located at the canonical carbohydratebinding surface, comparable to existing cellulose oligosaccharidebinding sites.

This work advances the functional and structural understanding of microbial expansins, highlighting how they modulate biomass and interact with ligands. Although the precise molecular mechanism underlying the disruption of noncovalent interactions in the plant cell wall remains unresolved, the study establishes a solid foundation for future research aimed at exploiting expansins for improved lignocellulose utilisation.