| Biomass cellulose is one of the most abundant renewable resources on the earth,and its efficient utilization could not only reduce the environmental pollution,but also alleviate the global energy crisis.Biomass resources can be used for the production of bioethanol,fiber materials and other high value-added products through biorefinery technology,among which the bioethanol production is considered to be the most promising development direction.While the biorefinery of biomass must rely on the biocatalysis and hydrolysis of cellulase to hydrolyze the biomass into platform sugars,which were further fermented by the microbial.However,the current study of cellulase were limited due to the poor productivity of strains,incomplete enzyme secretion,low hydrolysis efficiency for cellulosic substrates,high cost of cellulase production and incomplete regulation mechanism of cellulase synthesis and metabolism.Heavy ion beam irradiation has unique physical and biological advantage in the breeding of strains due to its high LET and RBE,high mutation rate and wide mutation spectrum.Therefore,the wild strains of T.longibrachia were irradiated by the heavy ion beams for mutation breeding,and the carbon source optimization and biomass degradation of cellulase produced by mutants were performed,then the analysis of transcriptomics and proteomics were performed to explore the high-yield mechanism and regulation process of cellulase.The main results of this paper are as follows:(1)Heavy ion beam mutagenesis and high-yielding strains breeding of T.longibrachiatum.The fungal strain was identified as T.longibrachiatum by molecular means on the basis of ITS sequences(ID:MW193401).The T.longibrachiatum LC strains were irradiated by the heavy-ion beams and its survival rate decreased from 84.77% to 16.65% with the dose increased from 40 Gy to 400 Gy.Two excellent mutant strains,LC-M4 and LC-M16,were obtained by the plate primary screening and shake flask re-screening.The comparison of fermentation between wild strain and mutant strains showed that the cellulase and xylanase activities of the two mutants were significantly improved,and the FPA reached 4.51IU/m L and 4.16 IU/m L,which were 46.91% and 35.5% higher than the wild strain,respectively,which work provides the new strain resources for the cellulase production on industrial scale.(2)Study on carbon source optimization for enzyme production by T.longiflorum LC-M4 mutant.The waste paper(waste office paper(WOP),tissue paper(TP),magazine paper(MP)and corrugated board(CB))were used for cellulase production and the FPA,CMC,?-glucosidase and xylanase activities reached 2.97 IU/m L,4.8IU/m L,0.51 IU/m L and 382.59 IU/m L using CB,respectively,which results showed that the CB could be used for the enzyme production by LC-M4 as carbon source.The structural characterization revealed that the main component of waste paper is cellulose,and the fillers such as calcium carbonate could provide trace elements for bacterial growth and product synthesis,which could promote the fermentation process.In order to further improve the enzyme activity,the CB and wheat bran(WB)were selected as mixed carbon sources(3:1/w:w)for enzyme production,and the maximum?-glucosidase of 0.80 IU/m L were obtained,which were significantly increased by56.86% when compared with the CB as single carbon source.In addition,the pNPCase,FPA and xylanase activities from the mixed carbon source have also been improved,indicating the CB and WB could be efficiently utilized as inductive feedstock for cellulase production by T.longiflorum,which have the great potential for industrial applications.(3)Study on the degradation process and mechanism of sweet sorghum straw(SSS)by cellulase produced by LC-M4 mutant.The enzymatic hydrolysis of SSS were significantly improved in 2% Na OH pretreatment and achieved 86.44%.The structural characterization revealed the destruction of lignin-polysaccharide interactions and increase of porosity and crystallinity after pretreatment.During the course of hydrolysis,the cellulase dissolved the cell walls firstly occurred from the middle of cell walls and then toward the cell wall corners.In addition,the vascular bundles in natural SSS were highly lignified,while the dilute alkali pretreatment can effectively delignification,thus improving the enzymatic hydrolysis efficiency of cellulose substrate.These results will help to design and modified the plant cell structure at the organization level aimed at reducing the recalcitrance of substrate,thereby improving the enzymatic hydrolysis efficiency of biomass cellulose.(4)Study on the high-yielding mechanism and secretion regulation process of cellulase by T.longiflorum mutants.The analysis of transcriptomics and proteomics of wild strains LC and mutant strains LC-M4 and LC-M16 showed that the protein processing in ER involved in protein secretory pathway,starch and sucrose metabolism pathway and N-Glycan biosynthesis pathway were significantly changed both in cellulase hyper-producing mutants,which were the main reason for the high yield of cellulase.In addition,the key candidate genes Sec61,PDI,VIP36,OST that affect the cellulase synthesis and secretion were obtained through coalition analysis,which could be used the targets of design and modification through genetic engineering of strains.In addition,a model of the secretion pathway of cellulase protein was constructed on the basis of the DEG/DEP and key candidate genes obtained in this study,which supplemented the process of cellulase metabolism regulation and synthesis secretion pathway of filamentous fungal. |
PDF Full Text Request