The recent discovery of the carbohydrate-active enzymes lytic polysaccharide monooxygenases (LPMOs) has greatly expanded our knowledge of lignocellulose degradation in nature. The application of LPMOs in industrial enzymatic cocktails has had a crucial influence on the efficiency of biomass saccharification for biofuels. In biology, LPMOs are widely distributed across the taxa and have been proposed to play a role in the first wave of attack of various pathogens. A large number of plant and human pathogens contain LPMOs in their genomes. Therefore, in addition to their industrial utilization, the biological role of LPMOs has become a topic with increased scientific interest.
Despite developments in the field that implicate LPMOs in pathogen virulence, the topic of how to control their activity has remained largely unexplored. With no prior studies on the topic, this PhD project investigates LPMO inhibition by natural extracts. The investigation focuses on plants as a source of LPMO inhibitors, due to plants’ innate ability to produce defense compounds against microbial pathogens. Furthermore, the work investigates central questions related to LPMO activity and synergy.
Improving the fundamental understanding of LPMOs is important from a scientific perspective, as well as for the enzymes’ optimal industrial applications. The PhD thesis first sets out to investigate LPMO activity mechanism and synergy with other cellulolytic enzymes. Paper I presents the results from an investigation with focus on cellulaseLPMO synergy. Overcoming cellulose
recalcitrance through enzyme synergy is an important factor for the effective utilization of biomass. The synergy between three LPMOs and two cellulases was investigated systematically, providing important knowledge on how the enzymes’ mode of action can affect synergy. Using welldefined
enzyme preparations and robust kinetic assays, the paper advances our current understanding that LPMO regioselectivity is a key factor in synergy with cellulases of different directionality. The individual assessment of LPMOs is not only necessary for synergy studies, but also for LPMO activity mechanism, due to the documented interaction of LPMOs with oxygen and hydrogen peroxide. Therefore, the activity of the model LPMO LsAA9A, from L. similis, was investigated next. Results, presented in Paper II, show for the first time the ability of LsAA9A to utilize hydrogen peroxide for carbohydrate breakdown. Using robust activity assays for comprehensive LPMO characterization we present evidence of an orderedsequential reaction mechanism, which improves our understanding of LPMO catalysis.
The second part of the thesis explores the topic of LPMO inhibition by plant extracts. The hypothesis that plants may produce compounds with inhibitory properties towards LPMOs in particular is tested by utilizing nature’s diverse plant catalogue for the development of an extensive library of plant extracts. Plants have been utilized throughout the ages for the extraction of compounds with pharmacological and agricultural importance. In Paper III, plant extracts from the library were tested for LPMO activitymodulating properties against the model LPMO, LsAA9A. Inhibition screening lead to the detection of a number of plant extracts with high inhibitory potency, with a pronounced effect for methanolic plant extracts. Strong LPMO inhibition was observed for methanolic extract of C. cassia (cinnamon), which served as a focus for further investigations. Extensive characterization of the methanolic cinnamon extract, aided by protein crystallography and mass spectrometry, lead to the identification of the polyphenolic compound, cinnamtannin B1, as an LPMO inhibitor.
The current thesis contributes to our understanding of LPMO synergy and activity and sets the groundwork of natural LPMO inhibition. The results show for the first time the successful identification of a natural LPMO inhibitor, derived from a plant extract, which confirms the hypothesis that compounds can be found in plants that possess natural LPMO inhibitory properties. However, important questions remain open, such as the biological application of LPMO inhibitors. With recent reports on the importance of LPMOs for pathogen virulence, such questions become even more pertinent. Future research on the agricultural and pharmacological applications of such inhibitors may contribute to the development of tailored antimicrobial agents and pathogenresistant
crops.