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Plant biomass delignification is crucial for terrestrial carbon cycling and is essential for incentives aiming at the valorization of lignocellulose. For understanding this central process in nature and biorefinery, we need to elucidate and comprehend the mechanisms that govern recalcitrance and conversion at the molecular level. This insight can only be obtained by accurate analysis of the molecules involved, both contentwise and structurewise. This research, therefore, aimed to advance the quantitative mapping of lignin and employ the developed analytical toolkit to unravel the underlying mechanisms of a process that has largely remained elusive to date: the fungal delignification of plant biomass. We demonstrate through careful method validation that py-GC-MS analysis can be used for the concurrent quantification and structural characterization of grass, hardwood and softwood lignin, when employing uniformly 13C labeled lignin internal standards and relative response factors for the individual pyrolysis products. The developed method was used to assess three white-rot fungal species in terms of delignification effectivity and selectivity. In both important traits, Ceriporiopsis subvermispora outperformed Lentinula edodes and Pleurotus eryngii. Comprehensive structural analyses of the residual lignin after growth of C. subvermispora allowed us to reconstruct various degradation routes of lignin’s most abundant β-O-4’ ethers and determine the relative susceptibility of various β-O-4’ substructures. Our results imply that one-electron oxidation initiates in situ ligninolysis, which then cascades into the cleavage of Ca-Cb, Cb-O and O-4’-aryl bonds. Ligninolysis was shown to depend on the electron density of the 4’-O­-subunit, diastereochemistry and γ-acylation. In addition to white-rot basidiomycete fungi, we studied the ligninolytic capacity of the ascomycete fungus P. anserina. Substrate characterization unambiguously confirmed lignin degradation and secretome analysis suggested that laccases and H2O2 producing enzymes were likely involved in the observed ligninolysis.

Quantitative mapping of lignin : Comprehensive insight into fungal delignification of plant biomass