The Degradation Specificity Of Thermobifida Fusca Studied By Structural Bioinformatics And Functional Omics

Lignocellulose is the most abundant renewable biomass in nature.Degradation and conversion of lignocellulosic biomass is attracting attention because of its potential for the development of sustainable and environment-friendly energy.Given the natural recalcitrance of lignocellulo se to enzymatic hydrolysis,its efficient degradation depends on the synergistic effect of microorganisms and degrading enzymes.Therefore,it is important to understand the niche of specific microorganisms with high efficiency,and how the glycoside hydrolases and other auxiliary enzymes degrade substrate,which would be helpful for analyzing the microorganism's mechanism of efficient degradation and growth preference,and laying the theoretical foundation for the engineering new enzyme.The integration of omics and structure bio informatics technology provide a platform for the above analysis.In this paper,functional genomics was used to analyze the genome of Thermobifida fusca and its dynamic changes of extracellular proteome,and its growth preference and efficient degradation mechanism were elucidated.Based on structural bio informatics,the fundamental structure of enzyme to identifiy the substrate was analyzed,and the substrate-binding specificity and promiscuity help us propose a mode of synergistic degradation.The main results of this work as follows:(1)Ligand-binding specificity and promiscuity of the main glycoside hydrolase families as revealed by active-site architecture analysisBiomass can be converted into sugars by a series of lignocellulo lytic enzymes,which belong to the glycoside hydrolase(GH)families summarized in CAZy databases.Here,using a structural bioinformatics method,we analyzed the active site architecture of the main lignocellulolytic enzyme families,including cellulase,xylanase and ?-glucosidase.The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure.And the number of potential subsites was significantly different among different families.In the cellulase and xylanase families,the conserved amino acids in the active site architecture were mostly found at the-2 to +1 subsites,while in ?-glucosidase they were mainly concentrated at the-1 subsite.Families with more conserved binding amino acid residues displayed strong selectivity for their ligands,while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands.Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand.These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes.(2)Analysis of the oxidation mechanism of lignocellulolytic auxiliary enzymes by structural bioinformaticsLignocellulolytic auxiliary enzymes are an indispensable part of the complete degradation of lignocellulose.By studying the active region of the enzyme,it was found that the preferred amino acids in this region were His/Cys/Glu and Trp/Tyr/Phe,which have polar groups or benzene rings can assist electronic delivery.The acceptor of enzyme is far more selective than donor by reason of higher conservation of acceptor binding area than donor binding area.The amino acids responsible for electron transfer display a high degree of conservation.The amino acids responsible for electron transfer are either aromatic amino acids or polar amino acids.And the last amino acid for transfer electron to acceptor is usually His.In addition,based on the correlation between the electron donor and the receptor,the possible synergistic and competitive patterns between different enzymes were proposed,which provided the theoretical basis for the efficient synergistic degradation of the enzyme.In these enzymes,T.fusca only encodes the LPMOs of AA10,and its electron donor source has not yet been elucidated,so the efficient degradation of T.fusca can rule out factors ofAA10.(3)Analysis of Thermobifida fusca degradation potential from the genome The phylogenetic relationship and the number of lignocellulose-degrading enzymes between T.fusca and other 18 lignocellulose-degrading actinomycetes were compared.It was found that the number of CAZy gene in T.fusca is relatively less,so the type of substrate that can be completely degraded is limited.T.fusca has a comprehensive cellulase system,including lytic polysaccharide monooxygenase(LPMO),lacks side chain degrading enzyme of hemicellulose and lignin-degrading enzyme.In these enzymes,nearly 40%of the enzyme possess carbohydrate binding domain(CBM),and CBM2 has the highest frequency.(4)Effects of different substrates on Thermobifida fusca extracellular proteomeThe physical and chemical properties and the dynamic changes of the extracellular proteome of T.fusca were determined by using different lignocellulosic substrates.It was found that the optimum conditions for culture is cellulose and mannan,and the appropriate concentration of disaccharides as the inducers of main glycoside hydrolase secretion.The secretion of extracellular cellulose has more GH6 and GH9,less AA10,which in line with the efficient degradation mechanism of T.fusca speculated by structure bioinformatics.Besides,it was found that the inducible protein of cellulose and mannan was present on the cell membrane,which induced the partial degrading enzyme to be secreted first,and the subsequent disaccharide induced the major degrading enzyme.