Discovery and applications of family AA9 lytic polysaccharide monooxygenases

Doctoral thesis, 2023

Auxililary activity family 9 lytic polysaccharide monooxygenases (abbreviated as AA9s or LPMO9s) are fungal mono-copper enzymes capable of oxidatively cleaving various plant cell wall oligo- and/or polysaccharides. LPMO9s are key components of lignocellulolytic enzyme cocktails used in today’s biorefineries to break down biomass into fermentable sugars.
Highly stable enzymes with novel functions are of great interest to improve enzymatic biorefinery processes and their economic feasibility. Genome sequencing of an industrially relevant fungus, Thermothielavioides terrestris LPH172, revealed 411 putative carbohydrate-active enzyme (CAZy) domains. Transcriptomic analysis indicated that the fungus upregulated numerous LPMO9 genes in concert with canonical cellulase and hemicellulase encoding genes to degrade lignocellulose. Nuanced co-upregulation was detected for LPMO9 genes and those encoding other redox-active CAZymes. Six strongly upregulated TtLPMO9 genes were heterologously expressed and functionally characterized using cellulosic and hemicellulosic substrates. These studies showed that the multitude of LPMO9 genes provided the fungus with different functions, including previously unknown cleavage of cellulose-associated spruce arabinoglucuronoxylan and acetylated birch glucuronoxylan. In a related study, xylanolytic LPMO9 activity was revealed or enhanced by debranching xylans enzymatically, which likely assumed a rigid and stretched xylan conformation that associated with cellulose to increase accessibility to LPMO9s.
LPMOs have unique oxidative powers which render them advantageous for various biorefinery applications. A C1-oxidizing TtLPMO9G was found to increase the amount of carboxyl groups on sulfated cellulose nanocrystals by 10%, without any extensive degradation of the crystals. The functional groups thus generated were used for proof-of-concept crosslinking, which could aid in the production of bio-based materials. In another application, addition of TaLPMO9A to a benchmark LPMO-poor cellulolytic cocktail was shown to improve saccharification yields of mildly pretreated spruce substrates. The final glucose and xylose yields were increased by up to 1.6- and 1.5-fold, respectively, illustrating how LPMO9s can be exploited in the saccharification of these notoriously recalcitrant substrates.