Insights Into The High-efficiency Lignocellulose Degradation Mechanism Of Chaetomium Thermophilum Based On Integrated Omics

Plant biomass is the most abundant and sustainable renewable resource on the earth.Therefore,improving the comprehensive utilization rate of plant biomass,producing high value-added products and forming a green and efficient transformation mode are the development demands for modern biological technology.The diverse composition and complex structure of plant cell wall make it difficult for the enzyme to degrade,called "biomass recalcitrance",and cellulose is the main component of lignocellulose.Crystalline cellulose with a dense structure has become the bottleneck in the process of biomass degradation.So the discovery and construction of a more efficient enzymatic system to degrade crystalline cellulose have become a research hotspot on biomass conversion.The discovery of lytic polysaccharide monooxygenase(LPMO)rewrote the research process of crystalline cellulose-degrading enzyme system in 2010.LPMO can act as "explosion-initiating agent" by breaking glucosidic bonds in an oxidation way,and improve the degradation efficiency of cellulose hydrolases rapidly.Thus LPMO has become a new research hotspot.Chaetomium thermophilum,a dominant thermophilic fungus in composting fermentation,is widely distributed in cellulose-rich and self-heating habitats with an optimal growth temperature of 50? and has a high ability to degrade cellulose Therefore,a systematic and comprehensive research is needed to study the biomass degradation preference of thermophilic fungi,temporal expression of enzyme system during biomass degradation and the synergy between different enzyme components.In this way,we can precisely customize high-efficiency enzyme cocktails to degrade specific substrates,which is of great significance to improve biomass conversion efficiency and sustainable development of economy.In this paper,the relative mechanisms of C.thermophilum were studied by using integrated omics methods such as genomics,transcriptional quantitative analysis,proteomics and structural bioinformatics.The main results are as follows:1.The composition of carbohydrate-active enzymes(CAZymes)in the genome of C.thermophilum showed that this fungus had a complete enzyme system of hydrolase and oxidase to degrade crystalline cellulose and preferred to degrade the cell wall of monocots.The CAZymes encoded by C.thermophilum was analyzed by genomics.The result showed that this fungus encoded 298 CAZymes,and genes related to the degradation of cellulose,xylan and ?-1,3;1,4-glucan were relatively comprehensive among them.So this fungus preferred to degrade the cell wall of monocots since cell walls of monocots were rich in xylan and ?-1,3;1,4-glucan.Further analysis found that enzymes related to crystalline cellulose degradation include 5 cellobiohydrolases(CBHs),8 ?-glucosidases(BGs),10 endoglucanases(EGs),18 AA9 LPMOs,2 cellobiose dehydrogenases(CDHs)and 8 glucose-methanol-choline oxidoreductases(GMCs),among which CDH and GMC could act as electron donors for LPMO.Therefore C.thermophilum has a complete enzyme system of hydrolases and oxidases to degrade crystalline cellulose.2.Using comparative proteomics analysis of C.thermophilum under different culture conditions identified that the fungus prefered to degrade the cell wall of monocots,and arabinose and MCC were two specific inducers for CAZymes.The relative content of AA9 LPMO was the highest when the fungus cultured on 50?.The dynamic changes of extracellular enzymes secreted by the fungus under complex natural substrate(wheat bran and corn bran),different culture temperatures,and 8 single pure carbons were detected over time,the results showed that corn bran(CB)could induce the fungus to secrete more extracellular protein and CAZymes,especially CBH and AA9 LPMO,compared with wheat bran(WB).It also showed that the secretion and dynamic changes of extracellular CAZymes,especially AA9 LPMO,by C.thermophilum,was closely related to temperature.Arabinose was a potent inducer among soluble sugar and it induced C.to secrete large amounts of xylanases,which was significantly different from other fungi that used xylose as a potent inducer.Insoluble microcrystalline cellulose(MCC),acting as a highly effective inducer,could induce the fungus to secrete the most amount of cellulose-degrading enzymes(reaching a relative content of 41%at day 5),the most species(12)and the highest relative content of AA9 LPMOs(13.8%).Two carbons,arabinose and MCC,had a obvious induction for CAZymes,especially AA9 LPMOs.It laid the foundation for further study of the mechanism of cellulose degradation and the novel induction of AA9 LPMO in C.thermophilum3.The temporal expression of extracellular CAZymes detected by transcriptional quantitative analysis and proteomics revealed the high-expression of LPM02 and the co-expression of LPMO2,CBH and CDH in C.thermophilum when cultured on MCC under high temperature.The specific carbon arabinose and MCC were used to conduct time-gradient cultivation of C.thermophilum,and proteome and RT-qPCR were used for dynamic tracking analysis.The results showed a definite temporal expression of CAZymes secreted by C.thermophilum under arabinose and MCC induction.Besides xylan-degrading enzymes,arabinose also induced the fungus to secrete more LPMO1 and LPMO3 with CBM1 modules in the late stage of cultivation.Significantly different from arabinose,the fungus could secrete a large number of cellulose-degrading enzymes under MCC induction,mainly including hydrolase-CBHs and oxidase-AA9 LPMOs.Meanwhile,an endoxylanase GOSBF1(GH10+CBM1)could be co-expressed with cellulases.What's more,LPMO2 could be induced specifically by MCC.The insert on loop3 in the substrate-binding surface expanded the interaction area between LPMO2 and substrate,which may be the reason why LPMO2 could efficiently bind to crystalline cellulose and catalyse under thermophilic conditions.The highly efficient co-expression of LPMO2s,CBHs and CDHs when C.thermophilum cultured on MCC under high temperature indicated a clear synergistic relationship between cellulose oxidase and hydrolase,which can guide the construction of the compact and efficient enzyme cocktails.4.The correlation analysis of the high-efficiency cellulose-degrading enzyme system based on integrated omics showed an obvious synergy expression of LPMO1 and CBH ?,LPMO2 and CBH ? under high temperature.Integrated omics was used for analysis when C.thermophilum cultured on MCC at a initial stage.The results showed an obvious correlation between CBH ? and LPMO2 in both transcription and protein expression levels,and the correlation between LPMO and CDH was higher than that of GMC,among multicomponent cellulases.It indicated the co-expression of CBH ?,LPMO2 and CDH,possibly having a same transcriptional regulatory mechanism.Moreover,CBHI and LPMO1 could also co-express.LPMO2,co-expressed with CBH ?,didn't have the CBM 1 module.This study detailed the synergy between different enzyme components under high temperature and found the co-expression of LPMO 1 and CBH I,LPMO2 and CBH ?.It lays foundation for further enriching and completing the degradation theory of crystalline cellulose and provides new ideas and solutions for the industrial application of enzyme cocktails.5.A LPMO2 of C.thermophilum was successfully cloned and heterologously expressed by using the yeast expression system.Enzymatic properties of LPMO2 were characterized,and an electrode analysis system of LPMO was preliminarily established.10 yeast expression plasmids of LPMOs were successfully constructed in this paper,and G0SH69,a LPMO2 protein,was successfully expressed and purified.Then a 2,6-DMP method was used to assay the activity of LPMO rapidly.The results showed that this LPMO could tolerate the temperature under 60?.It could be used as the electrode since it was an oxidase that could transfer electrons.So establishing the electrode method and electrochemical detecting system of LPMO is helpful for people to have a further study on the mechanism of LPMO,and provides a method and platform to have a research on substrate optimization,electron transfer and product forming efficiency.Clarifying the function of AA9 LPMO is helpful to elucidate the role of this enzyme in lignocellulose degradation process.