Study On Lignin Driving Lytic Polysaccharide Monooxygenases To Enhance Enzymatic Saccharification Of Cellulose

Utilizing lignocellulosic biomass for biorefinery was considered as a renewable and carbon-neutral development strategy,but the bioconversion process was significantly hindered by the resistant structure of lignocellulose itself.As the major lignocellulose decomposer in nature,white rot fungi can enhance lignocellulose hydrolysis by the secretion of a series of auxiliary activities(AA)enzymes.In this thesis,white rot fungus extracellular AA enzyme system was used to construct an efficient lignocellulose synergistic hydrolysis cocktail.Based on this,the hydrolysis enhancement mechanism of the key AA enzyme—lytic polysaccharide monooxygenase(LPMO)was clarified,and the mechanism of lignin in the oxidation reaction of LPMO was revealed.The main results of the research were discussed as follows:1)An efficient synergistic hydrolysis cocktail was constructed with secreted enzymes of white rot fungus.The extracellular enzyme system of 6 white rot fungi were evaluated for the effects of lignocellulose enzymatic hydrolysis.It was found that the secreted enzymes of E.taxodii2538 can significantly enhance the lignocellulose enzymatic hydrolysis by commercial cellulase.Under the optimal conditions,the synergistic hydrolysis cocktail that was constructed with the secreted enzymes of E.taxodii 2538 and commercial cellulase made the glucose yields of wheat straw and bamboo lignocellulose reach the highest 430.4 mg/g substrate and 329.3 mg/g substrate respectively,which were significantly increased by 12.1%and 20.4% respectively,compared with that by pure commercial cellulase.The enhancement of the secreted enzymes of E.taxodii 2538 on enzymatic hydrolysis was related to the content of lignin in lignocellulosic substrates,while the ligninolytic enzymes in the secreted enzymes of E.taxodii 2538 was not the key factor for hydrolysis enhancement.2)The key AA enzyme in the synergistic hydrolysis cocktail was identified.Transcriptome analysis of E.taxodii 2538 under three different culture conditions showed that when lignocellulose was used as the carbon source,the gene transcription levels of LPMO and cellobiose dehydrogenase(CDH)that drive LPMO reactions were significantly up-regulated.The up-regulation fold and transcription level of LPMO gene c17792?g1 and CDH gene c20079?g1 were much higher than other AA family genes.It suggested that LPMO might be the key AA enzyme for synergistic hydrolysis.The c17792?g1 gene was cloned and heterologously expressed to obtain the LPMO recombinant protein EtLPMO9 A.It was found that EtLPMO9 A had typical LPMO activity,and could significantly enhance the enzymatic hydrolysis of lignocellulose without adding extra electron donors.The cellulose conversion ratio of straw and bamboo lignocellulose were increased by 11.9% and 21.1%,respectively.And the enzymatic hydrolysis enhancement by EtLPMO9 A was positively correlated with the content of lignin in the substrate.The effects of EtLPMO9 A on lignocellulose hydrolysis was similar to that of the secreted enzymes of E.taxodii 2538,indicating that EtLPMO9 A was the key AA enzyme for hydrolysis enhancement.3)The mechanism that lignin in-situ drives LPMO to enhance cellulose hydrolysis was revealedA lignocellulosic mimicking model was constructed as substrate for the study,which was equipped with a polysaccharide template of self-assembled bacterial cellulose film and synthesized lignin.Remarkably,it was demonstrated that lignin polymer deposited on cellulose could reduce LPMO in-situ for cellulose oxidation and then boost cellulose hydrolysis.More importantly,lignin in-situ might exceed the well-known reductant of ascorbic acid to drive LPMO for lignocellulose enzymatic hydrolysis,with equivalent cellulose oxidation efficiency and 70.4% lower hydrogen peroxide generation,avoiding the inactivation of enzymes.In summary,based on the constructed synergistic system for lignocellulose enzymatic hydrolysis with the secreted enzymes of white rot fungus E.taxodii 2538,this research revealed the oxidative cleavage mechanism of cellulose in white rot fungus lignocellulose degradation system,which was catalyzed by lignin-driven LPMO,and further clarified the enhancement mechanism of lignocellulose enzymatic hydrolysis by LPMO.This study provided a new strategy for the efficient saccharification of lignocellulose,and the feasibility was demonstrated to efficiently saccharify lignocellulose without lignin removal by harsh thermochemical pretreatments,which provided new theoretical support for the development of lignocellulose biorefinery.