| Lentinula edodes is a delicious and nutritious edible mushroom.The major production of L.edodes is in China,accounting for more than 85% of the world yield.At present,hardwood cork oak(Quercus variabilis)is used as raw material for L.edodes cultivation.The current cultivation model consumes a great deal of forest resources,posing a threat to the environment.New materials are needed to substitute the wood as substrates.However,the mechanism of lignocellulose degradation by L.edodes is unclear,which limits the improvement of lignocellulose bioconversion and the development of new substrates.Our lab sequenced the genome of a L.edodes monokaryon,providing an opportunity to understand the lignocellulose degradation mechanism of L.edodes.1.To explore the profiling of soluble secreted proteins involved in lignocellulose degradation in L.edodes.For that,the secretomes of L.edodes grown on microcrystalline cellulose,cellulose with lignosulfonate(3 simple carbon sources)were compared to glucose.Based on nanoliquid chromatography coupled with tandem mass spectrometry of whole-protein hydrolysate,230 proteins were identified.Label-free proteomic analysis showed that the most abundant carbohydrate-active enzymes involved in polysaccharide hydrolysis were endo-?-1,4-glucanase,?-galactosidase,polygalacturonase and glucoamylase in both cellulosic secretomes.In contrast,enzymes involved in lignin degradation were most abundant in glucose culture,with LACC 1 being the predominant protein(13.13%).When the cellulose and cellulose with lignosulfonate secretomes were compared,the abundance of cellulases and hemicellulases was higher in cellulose with lignosulfonate cultures,which was confirmed by enzyme activity assays.In addition,qRT-PCR analysis demonstrated that the expression levels of genes encoding cellulases and hemicellulases were significantly increased(by 32.2-to 1166.7-fold)when L.edodes was grown in cellulose with lignosulfonate medium.The presented results revealed the profiling of extracellular enzymes involved in lignocellulose degradation.In addition,SLS raised the expression of genes encoding cellulases and hemicellulases.2.Comparative analysis of proteins secreted by L.edodes in woody and non-woody biomass reveals the natural lignocellulose degradation mechanism.Secretomes of L.edodes cultured in wood chips(woody),caragana,rice straw,maize straw,rape straw,corn cob and cottonseed shell(non-woody)media were compared.Based on the nano LC-MS/MS analysis,154 different proteins were identified in 7 secretomes.L.edodes secreted 78 proteins in rice straw medium,whereas,more than 100 proteins in other media.Among the 44 proteins shared in the 7 secretomes,CAZy protein accounts for about 60%,mainly including the cell wall polysaccharide degrading enzymes.This indicates that hydrolase system of L.edodes is widely adaptable to the degradation of polysaccharides in woody and non-woody biomass.However,ligninolytic enzymes including laccase(LACC13,LACC14,LACC12 and LACC9),class-II peroxidase MnP and glyoxal oxidase(AA5)were highly expressed in woody culture,less expressed or absent in other non-woody cultures,which suggested the lignin degrading enzyme system of L.edodes uniquely adapted to wood lignin;but at the same time,other strategies were used by L.edodes for degradation of lignin in non-woody biomass,such as CDH evolved in lignin degradation mechanism.3.Transcriptome analysis of lignocellulose degradation by L.edodes grown on glucose,cellulose and cellulose-SLS and screening candidate transcription factors of lignocellulose-degrading enzyme genes.A comparative transcriptome analysis was performed by L.edodes mycelia transferred to glucose,cellulose and cellulose-SLS media and cultured for 5 days,named as G5 d,C5d and CL5 d respectively.The results showed about 94% of CAZy genes were up-regulated in C5 d or CL5 d,4 of five differentially expressed transcription factor genes were up-or down-regulated in CL5 d,indicating that expression of plant polysaccharide degrading enzymes were regulated by SLS.GH6 and GH7-encoding genes were abundantly expressed in C5 d and CL5 d.Phylogenetic analysis based on protein sequences showed that GH6 and GH7 proteins were conserved,and meanwhile,L.edodes was closer to Polyporus in phylogenesis.In CL5 d,additionally,up-regulated non-CAZy genes included enzymes related to lignin degradation,such as cytochrome P450 monooxygenase,and enzymes involved in fatty acid metabolism.4.Comparative transcriptome analysis of the initial response to glucose and cellulose of L.edodes.A comparative transcriptome analysis was performed by L.edodes mycelia transferred to glucose(directly utilized carbon source)and cellulose(not directly utilized carbon source)cultured for a short time(0.5h and 2h)(named as G0.5h,G2 h,C0.5h and C2h).KOG enrichment analysis of genes specifically induced by glucose and cellulose showed that with 0.5h short-term stimulation,specific genes in response to glucose and cellulose distributed in many KOG classes;with 2h stimulation,the glucose responsive genes mainly gathered in primary metabolism and secondary metabolism classes,and cellulose responsive genes mainly in cellular processes and signaling classes.A detailed analysis of the genes in each KOG class showed that down-regulated genes were more than up-regulated genes in C0.5h,in contrary to genes in G0.5h.Furthermore,up-regulated genes were more than down-regulated genes until to 2h in cellulose medium.In summary,L.edodes secreted extracellular enzymes involved in lignocellulose degradation;sodium lignosulfonate could improve the expression of cellulase and hemicellulase genes.L.edodes was widely adaptable to the degradation of polysaccharides in woody and non-woody substrates.The lignin degrading enzyme system of L.edodes uniquely adapted to wood lignin,but L.edodes utilized other strategies to degrade lignin of non-woody biomass.Transcriptome analysis showed that sodium lignosulfonate regulated the expression of CAZy encoding genes.With 2h short-time stimulation,genes specificly in response to glucose distributed in the primary metabolism and secondary metabolic classes,and the genes specificly responsing cellulose were mainly in cell process and signal transduction classes.The above researches to explore the degradation mechanism of lignocellulose by L.edodes,are conducive to the utilization of new lignocellulosic materials according to enzyme system adaption of L.edodes and formula optimization;to assisting breeding and selection of strains with strong lignocellulose degrading ability;and to improving strains at the molecular level for the production of enzymes or biological conversion. |
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