| In the new century,crop straw as the inevitable agricultural waste is not fully utilized,it is usually burned in situ,which causes environmental pollution and resource waste.Whereas,high-efficient degradation of polysaccharide components in plant cell walls into biochemicals or biofuels is of great significance for sustainable development of green agriculture and industry in our country.Filamentous fungi is capable of secreting diverse lignocellulolytic enzymes,which is often utilized as the enzyme preparation for degradation of plant cell wall polysaccharides into fermentable sugars,however,due to the various evolutionary pathways and growth environments of plants,polysaccharide species and proportions in different kinds of plant cell walls show significant heterogeneity,thus in the coevolution process between microorganisms and plants,microorganism have evolved diverse degradation strategies,secreting different types and concentrations of enzymes,while lack of full understanding of the polysaccharide heterogeneity in plant cell walls and the microbial degradation preferences results in low utilization efficiency of plant cell walls and limits its large-scale application.Full study of the natural biomass hydrolytic preference in filamentous fungi will be beneficial for finding the efficient degradation enzymes for specific substrate and providing rational guidance for recomposing lignocellulolytic enzymes based on the type of substrates,in addition,it will provide theoretical support to improve the utilization efficiency of straw biomass.Lignocellulose hydrolytic preference of filamentous fungi mainly depends on the species and numbers of genes encoding lignocellulose degradation enzymes in the genomes,the transcription and expression of genes,as well as enzyme functional specificity,based on the background,this paper mainly takes three kinds of ascomycetous fungus especially Aspergillus niger as the research object and systemically studies its genome,proteome and extracellular polysaccharide degradation metabolome,the main results are as follows:1.Comparative genome analysis of the lignocellulose degradation enzymes discrepancy in ascomycetous fungus revealed the different degradation potential of fungiThe ITS sequence information of sequenced fungi including Aspergillus spp,Trichoderma spp,Neurospora spp,and Penicillium spp was obtained from JGI database and NCBI database,and the representative strains were selected to construct phylogenetic evolution tree,in addition,all the functional annotation proteins in each species were downloaded from the UniProt database,according to the structure of lignocellulose biomass and glycosidic bond types,the annotated proteins were classified and analyzed,results showed that in the genomes of different genus,Aspergillus spp especially Aspergillus fumigatus,Aspergillus niger,Aspergillus oryzae,Aspergillus flavus and Aspergillus nidulans possess relative higher types and numbers of lignocellulose degradation enzyme encoding genes,and in the Penicillius spp fungi genomes,less genes encoded xyloglucan and galactomannan degrading enzymes,xyloglucan and galactomannan mainly distributed in dicotyledonous plants,therefore,it is speculated that its ability to degrade dicotyledonous plants was not competitive,Trichoderma spp and Neuro,spora spp are located in in the same evolutionary branch,and both genus have less hemicellulase and pectinase but higher cellulase,which may result from lignocellulose degradation enzyme genes specialized for cellulase genes,making it obvious advantage in cellulose degradation,gene specialization phenomenon may be the foundation of filamentous fungi hydrolytic preference.In addition,the proportion of isozymes distributed in different families such as GH11 and GH10 family xylanases varied among species,therefore,the distribution difference of isozymes with different substrate specificity also leads to distinct biomass degradation ability of filamentous fungi.2.Comparative secretome analysis of the Aspergillus niger,Trichoderma reesei and Penicillium oxalicum under solid-state fermentation displayed their hydrolytic preferences for dicotyledon and monocotyledon plants polysaccharideThe composition and concentration of the extracellular enzymes secreted by Aspergillus niger,Trichoderrma reeseiand Penicillium oxalicum under solid-state fermentation were detected over time,the results showed that a large number of polysaccharide glycoside hydrolases were induced.With LC-MS/MS,a total of 279,161,183 proteins were detected in the secretomes of Aspergillus niger,Trichoderma reesei and Penicillium oxalicum,respectively,the results revealed that the extracellular proteins of Aspergillus niger were mainly for the degradation of polysaccharides in dicotyledonous plants such as galactomannan,xyloglucan and pectin backbone,and the extracellular enzymes of Penicillium oxalicum mainly involved in degradation of xylan and P-(1,3;1,4)-D-glucan distributed mostly in monocotyledon,and Trichoderma reesei mainly secreted cellulase and auxiliary proteins such as Cipl,Cip2,Swollenin etc,whereas relative to Trichoderma reesei and Aspergillus niger,the composition of cellulases secreted by Penicillium oxalicum is more balanced.This study further displayed the differences of extracellular functional proteins secreted by filamentous fungi in degradation of specific substrate at proteome level,which provide important guidance for exploring the degradation preferences of other microbes and recomposing enzymes rationally in industrial.3.Quantitative characterization different action modes of specific xylanases with FACE and LC-IT-TOF MS and establishment of extracellular polysaccharide degradation metabolomeUsing anion exchange chromatography and E.coli heterologous expression system,two GH10 family xylanases and five GH11 family xylanases were obtained,and FACE method was established to detect the heterogeneous xylan degradation products released by purified xylanases,results showed that this method was capable of accurately distinguishing the substituted XOS and unsubstituted XOS among heterogeneous xylan degradation products,in addition,the dynamic changes of different types of XOS over time were able to be quantitatively characterized.Based on quantitative analysis of the changes of different species of XOS over time,we found that GH10 and GH11 family xylanases were able to rapidly degrade xylan into a large number of XOS,while further degradation of XOS was slow,additionally,the smallest XOS released by GH10 and GH11 family xylanases in degrading substituted xylan backbone were identified as MeGlcA2Xyl3 and MeGlcA2Xyl4,respectively.Therefore,the two family xylanases have family specificity in degrading xylan backbone with substituents,whereas,in degradation of xylan backbone without sidechains,both of the two family xylanases can produce monosaccharide,xylobiose,xylotriose without family specificity,and the different species and concentrations of XOS produced by various enzymes mainly depended on the different binding force of subsites in enzyme active center,moreover,?-1,4-xylosidase was able to further degrade MeGlcA2Xyl4 into MeGlcA2Xyl3,while continuing degradation of MeGlcA2Xyl3 required side-chain degrading enzymes.The dynamic degradation products changes of pure enzymes displayed that high efficient degradation of heterogeneity polysaccharides required efficient sequential expression of backbone,side-chain degradation enzymes and glycosidases.4.Detection of dynamic changes of functional proteomics of Aspergillus niger grown on limited carbon sources proved substituted XOS play an important role in activating xylan metabolic systemA.niger An-76 is capable of degrading xylan completely and efficiently,using functional proteomics and qPCR,the extracellular,intracellular and membrane proteomes of A.niger An-76 grown on xylan-based substrates were systematically studied,the results demonstrated that A.niger An-76 was capable of secreting all the components of isozymes required for xylan complete degradation in a sequential order.Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars,which efficiently produced large amounts of XOS and xylose;however,XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR,resulting in higher xylanase activity and shortening xylanase-production time.Moreover,the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes,specific transporters,and key reductases anddehydrogenases in PCP.Therefore,it might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan(XOS)to cultures of filamentous fungi... |
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