High resolution product profiling of lytic polysaccharide monooxygenases

Abstract

Biorefinery of non-food lignocellulosic plant biomass to be valorized as biofuels, biochemicals and biomaterials is considered as a sustainable alternative for fossil resource-based production. In biorefinery, a key step is enzymatic degradation of plant cell wall polysaccharides into (fermentable) monomeric building blocks, which is typically driven by (hemi-)cellulases and essential lytic polysaccharide monooxygenases (LPMOs). Although some LPMOs have already been incorporated into industrial enzyme cocktails, only a limited number of LPMOs have been biochemically characterized. Therefore, this PhD thesis aimed to further shed light on the mode-ofaction, regioselectivity, substrate cleavage profiles and specificity of AA9 LPMOs from the fungi Myceliophthora thermophila C1 (MtLPMOs) and Neurospora crassa (NcLPMOs).We demonstrated that AA9 LPMOs show distinct cleavage profiles towards different types of cellulose; from bacterial cellulose mainly oxidized DP2-4 products were generated, while from the regenerated amorphous cellulose larger oxidized products prevailed. We developed a new method, making use of NaBD4-reduction in combination with hydrophilic interaction chromatography-mass spectrometric analysis, for separation and identification of LPMO-generated non-, C1-and C4-oxidized oligosaccharides. We found that oxidized (and reduced) oligosaccharides differ in their mass spectrometric fragmentation behaviors and patterns. Using this developed methodology, we discovered a series of novel double, C4 and C6, oxidized cellooligosaccharides. Again using this methodology, we identified and distinguished for the first time (isomeric) LPMO-oxidized xylogluco-oligosaccharides, and characterized two distinct oxidative xyloglucan degradation profiles. These xyloglucan substitution tolerant and intolerant cleavage profiles were correlated to specific active site segment configurations of AA9 LPMOs. Additionally, the active site segment configuration predicted for two (other) MtLPMOs, confirmed the aforementioned correlation with their xyloglucan substitution (in)tolerant cleavage profiles. Lastly, we studied two novel AA16 copper-dependent oxidoreductases, which were predicted to function as LPMOs. We showed their inactivity towards carbohydrate substrates, hence, disproved them being LPMOs, but we discovered that these AA16s can boost AA9 MtLPMOs to oxidatively degrade cellulose. Table of contents Chapter 1 General introduction Chapter 2 Oxidized product profiles of AA9 lytic polysaccharide monooxygenases depend on the type of cellulose Chapter 3 Mass spectrometric fragmentation patterns discriminate C1-and C4-oxidized cellooligosaccharides from their non-oxidized and reduced forms Chapter 4 Regioselectivity Substrate specificity MtLPMO9A 1 M. thermophila C1 No C1/C4 Cellulose (RAC), xylan associated to cellulose, xyloglucan 10 , mixed β-(1→3, 1→4)-linked glucan 10 MtLPMO9B 2 M. thermophila C1 CBM1 C1 Cellulose (RAC) MtLPMO9C 3 M. thermophila C1 No C4 Cellulose (RAC), xyloglucan 10 , mixed β-(1→3, 1→4)-linked glucan 10 ...