Research On The Molecular Mechanism Of Lignin Degradation By Irpex Lacteus CD2

Lignocellulose is one of the most abundant renewable resources on earth that exhibits great potential for biomass energy,but the lignin barrier limits enzymatic degradation of cellulose and hemicellulose during the transformation of lignocellulosic biomass into fermentable sugars,becoming the major bottleneck problem for efficient utilization of lignocellulose.White rot fungi are considered as the best lignin degraders,which could completely mineralize lignin into water and carbon dioxide through ligninolytic enzyme system and free radical system.However,little investigation was reported about how the ligninolytic enzyme system and free radical system of white rot fungi grown on lignocellulose and lignin worked.Thus,to elucidate underlying molecular mechanism involved in lignin degradation by white rot fungi under different conditions has great significance for improving biomass saccharification efficiency.This research selects the white rot fungus Irpex lacteus CD2 as the research object and reveals the molecular mechanism of lignin degradation by genomic,transcriptomic and biochemical analyses.The main results of the research are as follows:1.Genome sequencing and analysis reveals the lignin degrading system in I.lacteus CD2.We sequenced the genome of I.lacteus CD2 using a combination of Illumina HiSeq2000 platform and PacBio RS platform.Lignocellulolytic enzymes and other proteins involved in important biological processes were annotated.The near-complete genome sequence of I.lacteus CD2 was 43.2 Mb encoding 10,853 predicted genes.Among which273 carbohydrate active enzymes and 62 ligninolytic oxidoreductases genes were encoded.Manganese peroxidases?MnPs?,dye-decolorizing peroxidases?DyPs?and lignin peroxidases consisted of the main ligninolytic enzymes in I.lacteus CD2.Besides,the lignocellulolytic enzyme-encoding genes were not randomly distributed but tended to cluster in the I.lacteus CD2 genome.Phylogenetic analysis indicated that none of these adjacent genes appeared to be paralogs,indicative of frequent gene duplication events during evolution of I.lacteus CD2.2.Transcriptomic and biochemical analyses of I.lacteus CD2 grown on lignocellulose reveal a central role for manganese peroxidase in lignin degradation.The extracellular enzyme activities analysis of I.lacteus CD2 grown on lignocellulose showed that MnPs were rapidly induced,reaching maximal activity of158.1 U/L on the third day.Comparative transcriptome analysis exhibited that the transcript level of MnPs genes were two to three orders of magnitude higher than other ligninolytic enzymes.Meanwhile,genes involved in free radical generation were also upregulated.This revealed that the oxidative degradation of lignin was mainly through the combined action of MnPs-centred ligninolytic enzyme system and free radical system in I.lacteus CD2.MnPs could not only efficiently oxidize phenolic compounds in the absence or presence of Mn²⁺,but also generated carboxylate derived radicals for the oxidation of predominant non-phenolic lignin structures.The free radical system consisted of hydroxyl radical produced from Fenton reaction and superoxide anion radical,especially for carboxylate derived radicals generated from Mn³⁺and oxalate.Furthermore,various low molecular weight compounds including heme,oxalate,veratryl alcohol and hydrogen peroxide were involved in stimulating ligninolytic enzymes production and the ability to depolymerize lignin.In addition,iron transportation and iron reduction related to Fenton reaction promoted the generation of hydroxyl radical.3.Transcriptomic and biochemical analyses of I.lacteus CD2 grown on lignin reveal an important role for dye-decolorizing peroxidase in lignin degradation.Based on the extracellular enzyme activities and secretome analysis,I.lacteus CD2rapidly secreted DyPs,reaching maximal activity of 41.1 U/L on the fifth day.Moreover,the comparative transcriptomic analysis of I.lacteus CD2 grown on lignin was to further elucidate the molecular mechanism of lignin degradation.The transcriptomic analysis showed that the transcript level of DyPs genes were one to two orders of magnitude higher than other ligninolytic enzymes.However,there was no significant difference in the expression of genes associated with free radical generation.This revealed that the oxidative degradation of lignin was mainly through the action of DyPs-centred ligninolytic enzyme system in I.lacteus CD2.Furthermore,the biochemical analysis discovered that DyPs were able to catalyze the oxidation of phenolic lignin compounds as well as non-phenolic lignin compounds.More importantly,DyP enhanced the oxidation of non-phenolic lignin compounds in the presence of the mediator 1-hydroxybenzotriazole,which was similar to the laccase mediator system.In summary,based on genomic,transcriptomic and enzymatic analyses,this research systematically elucidates composition and expression regulation of ligninolytic enzyme system,free radical system and related metabolic pathways in I.lacteus CD2 grown on lignocellulose and lignin.The research achievements provide important information for further understanding molecular mechanism and regulation of lignin degradation by white rot fungi.