Synergistic Mechanism For Lignocellulose Degradation By A Thermophilic Fungus And A Thermophilic Actinobacterium Based On Functional-omics Analysis

Lignocellulose is the most abundant renewable resource on earth.The high-efficient degradation of lignocellulose is of great significance for sustainable development.However,lignocellulose with multi-layered compact structures constitutes the biomass recalcitrance.In natural habitats,relatively efficient microbial consortia and their degrading-enzyme systems have been formed over a long period of time,but their efficiency still cannot meet the high-efficient degradation requirement of lignocellulose conversion.Therefore,it is of great importance to understand the relationship between lignocellulose different-layered components and microbial enzymes,to discover the regulating characteristics of lignocellulosic structure and microorganisms as well as their enzymes,and to locate the key factors hampering high-efficient degradation of lignocellulose for improving the degrading efficiency of lignocellulose and further forming the environmental benign transformation process.Previous omics studies have reported that natural lignocellulose utilization systems are usually composed of microbial communities rather than a single microorganism.But it is still not clear that how these microorganisms perceive the structural layers of insoluble lignocellulose and how they coordinate with each other and form efficient synergistic degradation mechanisms.In addition,thermophilic microorganisms and thermostable enzymes exhibit significant advantages in industrial applications.Therefore,having a full insight into these thermophilic microorganisms,and constructing artificial high-temperature microbial consortia to perform the high-efficient degradation of lignocellulose have become a new hot area of researches.This paper takes the dominant functional microorganisms in high-temperature compost as research object,and mainly focus on their enzyme composition,degradation preference and capacity of utilizing reducing sugars as well as the possible bottleneck of lignocellulose degradation.The aim is to understand the synergetic mechanism and the key regulatory factors among high-temperature microbial consortia.The main results are listed as following:1.Analyzing the lignocellulose degradation potential of the representative thermophilic microorganisms in high-temperature ligincellulose composts based on comparative genomicAs the dominant fungus in the high-temperature lignocellulose composts,the genome size of Thermomyces lanuginosus is 19.16 Mb,which is 1/3 smaller than that of other filamentous fungi,and it only encodes 52 lignocellulose degradation related proteins.Abundant hemicellulose-degrading enzyme genes are detected in the genome of T.lanuginosus,and they are mainly ?-1,3;1,4-glucan and mannose oligosaccharide degradation related proteins.No exocellulases and endoglucanases are encoded in the genome of T.lanuginosus,but there are some lytic polysaccharide mono-oxygenases(LPMOs)genes.Thermobifida fusca is a thermophilic actinobacterium,and its genome encodes 27 lignocellulose degradation related proteins.Among them,the most abundant proteins are the cellulose-degrading enzymes,including 2 exocellulases,8 endoglucanases,3 ?-glycosidase and 2 LPMOs.T.fusca also encodes many xylan-degrading enzymes,including 3 endoxylanases,1 xylosidase and 3 xylan side-chain cleaving enzymes.In addition,T.fusca encodes 5 other lignocellulose-degrading enzymes.2.Using functional proteomics to characterize the substrate degradation preference and reducing sugar utilization ability of T.lanuginosusFunctional-omics method was used to analyze the reducing sugar utilization ability and the secretome of T.lanuginosus grown on 13 carbon sources.The results showed that T.lanuginosus preferred to utilize soluble sugars for growth.Among the 13 identified glycoside hydrolases with high expression levels,9 were reduced sugar glycosidases(RSGs),and 7 of the 10 identified proteases with high expression levels were exopeptidases.These results indicated that T.lanuginosus could utilize oligosaccharides and oligopeptides in saprophytic habitats for growth.Glycan-degrading enzymes of T.lanuginosus were specifically induced by xylan substrate,and T.lanuginosus mainly secreted one GH11 family xylanase that could degrade xylan to soluble xylo-oligosaccharides(XOS),in which unsubstituted XOS were rapidly absorbed and utilized by T.lanuginosus.However,T.lanuginosus did not secrete xylan side-chain cleaving enzymes,thus the substituted XOS(SXOS)could not be degraded and utilized by T.lanuginosus,which leaded to the prebiotics SXOS accumulation and output made up more than 8%(w/v)of the total xylan.T.lanuginosus exhibits distinct advantages in utilizing cheap materials producing thermostable xylanase and the high value-added SXOS as well as microbial inoculants.Therefore,T.lanuginosus has the potential of industrial application promotion.3.Using functional proteomics to characterize the substrate degradation preference and reducing sugar utilization ability of T.fuscaThe functional-omics results showed that cellulose could induce the secretion of a series of cellulose-degrading enzymes(including 2 exocellulases,4 endoglucanases and 1 LPMO)by T.fusca,and it could use the cellulose-degrading products for growth.Xylan induced T.fusca to secrete a xylanase from GH11 family(with relative secretion of 8.71±3.83%)and a xylanase from GH10 family(with relative secretion of 4.5±1.23%),and they could rapidly degrade xylan to XOS and xylose within 2 min.However,when different concentrations of XOS and xylose were used as the carbon sources to cultivate T.fusca,it showed that the growth and enzyme production of T.fusca were significantly inhibited if the concentration of XOS and xylose was higher than 0.5%(w/v).It exhibited that T.fusca genome encodes xylan-degrading enzymes which could be secreted to its secretome,while the high-concentration XOS and xylose produced by xylan degradation obviously inhibited the growth of T.fusca.In other words,T.fusca possesses efficient xylan degradation enzymes system,but lacks the efficient xylan-utilizing ability,which maybe the reason of T.fusca could not grow alone on natural maize straw.Therefore,T.fusca as the dominant actinobacterium in high-temperature composts,it should have synergistic relationships with other thermophilic microorganisms in natural maize straw composts.4.The functional-omics were used to reveal the synergistic mechanism for lignocellulose degradation by T.lanuginosus and T.fusca,demonstrating that the concentration of xylan-degrading products regulated their synergyMixtures of cellulose and xylan degradation products with different concentrations were used as the carbon sources to cultivate T.lanuginosus and T.fusca,and their growth and enzymes production as well as their synergistic mechanism were further analyzed.T.lanuginosus could rapidly utilize high-concentration XOS,thus it may be beneficial to remove the inhibition of high-concentration XOS to T.fusca.T.lanuginosus and T.fusca were used to construct an artificial microbial consortium,the results showed that T.lanuginosus grew rapidly in the initial stage and mainly degraded the insoluble xylan in the outer layer of lignocellulose by secreting a GH11 xylanase.While T.grew rapidly in the late stage and secreted abundant cellulases and xylanases.The xylanases could degrade lignocellulose to XOS and xylose which supported T.lanuginosus growth,thus promoting to expose the inner cellulose.T.fusca then secreted cellulases to degrade exposed cellulose and quickly utilized the cellulose-degrading products for growth.There was a negative correlation between the response of T.lanuginosus and T.fusca to different concentration of xylan-degrading products,that is,high concentration of XOS promoted the growth of T.lanuginosus but inhibited the growth of T.fusca.The low concentration of XOS is the signal of hemicellulose reduction and insoluble cellulose exposure in lignocellulose,thus promoted the growth of thermophilic actinobacterium.Therefore,the concentrations of xylan-degrading products especially the concentration of XOS in the habitat,regulates the growth and enzyme production of microorganisms with different substrate degradation preferences,which is the result of long-term evolutionary adaptation of the microbial consortia.This study constructed a microbial consortium containing thermophilic fungus and actinobacterium grew at 55?,and identified that the concentration of XOS is a key factor to regulate their growth,which provided a new idea for the artificial construction of other high-temperature and high-efficient lignocellulose-degrading microbial consortia.5.Revealing the function of GH10 xylanase which coexpressed with cellulases based on transcriptional quantitative analysis and protein heterologous expressionQuantitative PCR was used to detect the transcriptional expression levels of the important glucosidase hydrolases of T fusca induced by different substrates,and especially heterologous expressed a specific GH10 xylanase which coexpressed with cellulases.The results showed that xylan specifically induced the secretion of a GH11 xylanase Xyl11A by T.fusca,while cellulose specifically induced the secretion of the GH10 xylanase Xyl10A.Xyl10A was co-expressed with cellulases induced by cellulose,and Xyl10A contains a CBM2 which is mainly binding to crystalline cellulose,indicating that Xyl10A is an enzyme that binds to cellulose surface and degrades xylan.The optimal temperature of Xyl10A is 80? and the optimal pH of Xyl10A is 9,indicating that Xyl10A is a thermotolerant and alkaline-tolerance xylanase,thus it has an advantage to be used in pulp bleaching industry.6.Structural bioinformatics analyzing the structural basis of T.fusca xylanase Xyl10A interacted with ligand and the modification direction of improving its enzymatic activityThe interaction mechanism of Xyl10A active-site architecture amino acid residues and ligand was explored based on the structural bioinformatics analysis.The active-site architecture subsites are from-3 to+2 for GH10 proteins,which contains a total of 18 amino acid residues interacting with xylose rings at these subsites,among which 12 amino acid residues with high conservation locate at the-2 and-1 subsites After mutating each of these amino acid residues to alanine,the binding ability and relative enzyme activity of these mutants were significantly decreased.It indicated that these amino acid residues at-2 and-1 subsites were the main functional areas of GH10 family enzymes to efficiently bind substrates.The xylose ring at+1 subsite is basically not involved in the recognition and binding of functional residues,thus this subsite can contain substituent xylose ring,which is the structural basis for GH10 family xylanases to efficiently degrade substituent xylan.It is worth noting that the relative enzyme activity was significantly increased by over 90%when the amino acid residue 51E,which is interacted with the distal-3 subsite xylose ring,was mutated to alanine.It indicated that the modification direction of GH10 family enzymes is reducing the binding force of amino acid residues with distal subsite xylose ring,which is obviously different from that of the GH11 family enzymes to increase binding force of the distal subsite residues to substrate.This study has preliminarily elucidated the structural characteristics of GH10 xylanases interacted with ligand,and provided a direction for the further modification of proteins in this family.