Mechanism On Overcoming Biomass Recalcitrance In Wheat Straw By Pleurotus Ostreatus Through Deconstructing Cross-linked Structure

The efficient release of sugars in biomass was limited by biomass recalcitrance,which resluts from complex structures of plant cell wall,impeding low cost biorefiny of biomass.To develope efficient biorefiny technology of biomass,two key points should be clarified:first,the connection of cell wall structures with biomass recalcitrance;second,how to overcoming the key resistant structures.Pleurotus ostreatus BP3 can remove the resistance barrier of wheat straw and significantly enhance the enzymatic transformation of wheat straw,but the key factor and molecular mechanism for removing the resistance barrier are not clear.The analysis of the substrate structure and proteomics analysis were carried in the research.First,based on quantitative analysis method for analyzing the structure of lignocellulose after biological pretreatment,cell wall structure of wheat straw by P.ostreatus BP3 treatment were analyzed to clarify the key factor of removing the barrier of enzymatic resistance.Then,the molecular mechanism of P.ostreatus BP3 to relieve the resistance barrier of wheat straw was further clarified by secretory proteomics analysis,and lytic polysaccharide monooxygenases(LPMO)played a important role.Finally,the upexpressed LPMO9 B was expressed heterologously,and its function in the degradation of non-polysaccharides,especially the lignin-carbohydrate complex(LCC)bonds was revealed through a series of biochemical experiments.It explained how LPMO catalyzed iron reduction and hydroxyl radicals generation,expanding the function of LPMO and provides new ideas and guidance for the bio-refinery of wheat straw.The main results of this thesis are as follows:The accuracy of cell wall HSQC(Heteronuclear single quantum correlation)used in wheat straw structure analysis was verified.Combining with methoxyl determination,cell wall HSQC provided accurate semi-quantitative information of wheat straw structures.With easy process and low consuption,cell wall HSQC was a suitable method to analyze the structure of lignocellulose after biological pretreatment.After P.ostreatus BP3 treatment,enzymatic hydrolysis of wheat straw resulted in the cellulose conversion rate increased from21.3% to 93.2%,which indicated that the resistance barrier for enzymatic hydrolysis was effectively removed.Combined a semi-quantitative analysis method of complete component HSQC and methoxyl determination,the structure of the treated wheat straw was analyzed.It was found that the key factors for removing the resistance barrier were the degradation of lignin and the breaking of the lignin-carbohydrate bond.The lignin content is significantly negatively correlated with the cellulose conversion rate.P.ostreatus BP3 can break the lignin unit structure by splitting the carbon-oxygen and carbon-carbon bonds,which led to decreasement of lignin content,change of lignin structure and reduction of the resistance for enzymatic hydrolysis.The ferulic acid group and the ferulic acid ester bond play a role in bridging lignin and hemicellulose in the wheat straw.P.ostreatus BP3 can break the ferulic acid ester bond and deconstruct the structure of the cell wall,resulting in the reduction of the resistance to enzymatic hydrolysis of wheat straw.Based on the secretory proteomics,the protein expression of P.ostreatus BP3 was analyzed during the degradation of wheat straw.Versatile peroxidase(VP)and other Class II peroxidases are the main enzymes for P.ostreatus BP3 to degrade lignin.However,the up-regulated expression of VP was not the key in enhancing lignin degradation.Hydrogen peroxide-generating enzymes as auxiliary enzymes are the main factor for P.ostreatus BP3 to promote lignin degradation.The upregulated expression of LPMO could benefit for biomass recalcitrance removal.It speculated that the free radical system plays an important role in P.ostreatus BP3 to relieve the resistance barrier of wheat straw.The up-regulated expression of iron reduction-related protein promoted the occurrence of Fenton reaction and further enhanced the generation of hydroxyl radicals,which led to the degradation of lignin and LCC.Po LPMO9 B gene up-regulated during treatment of wheat straw by P.ostreatus BP3 was cloned and expressed heterologously in Pichia pastoris X33.Po LPMO9 B oxidized cellulose and enhanced the production of hydroxyl radicals by driving the oxidation reaction of hydroquinone and accelerating the reduction of iron.The generating hydroxyl radicals can effectively degrade and break LCC bonds to release ferulic acid,break lignin bonds,and oxidize lignin groups.Biochemical experiments revealed that Po LPMO9 B could drives the generation of hydroxyl radicals by enhancing the iron reduction reaction of the extracellular culture of P.ostreatus BP3.In summary,this study revealed the key factors of removing the resistance barrier of wheat straw based on the structural and molecular analysis.Non-polysaccharide degradation pathway and mechanism of LPMO involved in lignocellulose degradation was clarified.This research provides new ideas and guidance for the bio-refinery of wheat straw,broadens the functions of LPMO,and expands the understanding of the role of LPMO in the degradation of lignocellulosic biomass.