Regulation Of Extracellular Proteases And Improvement Of Cellulase Production In Trichoderma Reesei

Plant biomass is the most widely distributed and most abundant renewable resource on the earth.Biorefinery processes have been developed to produce biofuels and chemicals from the plant biomass feedstock and reduce human's dependence on fossil energy.In the process of biorefinery,lignocellulosic enzymes catalyze cellulose into fermentable sugars is the key step in determining the cost of biobased chemicals.The filamentous fungus Trichoderma reesei is the major fungal platform for industrial cellulase production.Although the secretion levels of extracellular cellulase from T.reesei at over 40 g/L,the cellulase activity during the late stage of the fermentation under the condition of cellulase production medium was significantly decrease.This reason may be related to the degradation of cellulase by extracellular proteases.On the other hand,the presence of proteases can hydrolyze the complex organic nitrogen into available nitrogen source for the strain growth and protein synthesis.Therefore,the contradiction between these two requirements prompted us to study the function of extracellular proteases in T.reesei.The expression of extracellular protease genes in Aspergillus sp and N.crassa were strictly regulated by several transcription factors such as AreA,XprG and PrtT.The ability of extracellular proteases secretion in T.reesei has been reported for a long time.However,the regulatory mechanisms is unknown in T.reesei.Therefore,in this study,we have studied the functions and regulatory mechanisms of secretory proteases in T.reesei and strain improvement.1.Identification and characterization of 12 proteases in T.reesei.The cellulase seriously degraded by secreted protease during the late stage of fermentation under the condition of cellulase production medium.Therefore,we identified the the secreted proteins from the fermented supernatant of 3d and 7d cultures using the proteomics.Using the bioinformatics analysis,12 of protease genes were obtained and subsequently deleted in T.reesei.We have found that deletion of the Tr123244 resulted in fewer conidia and lower cellulase production compared with that in QM9414?mus53 and the remaining 11 protease deleltion strains have the normal phenotype.Through analysis the proteolytic ability and protease activity in protease-deletion strain,we have found that ?Tr80170,?Tr120998 and ?Tr123234 resulted in the decrease extracellular protease activityies.Using hph resistant marker,we successfully knocked out proteases Tr80170,Tr120998 and Tr123234 by one step manipulation and constructed the three-protease genes knockout strains ?P57 and AP70.The skim milk plate shows that the proteolytic abilities were significantly decreased in AP57 and AP70.The extracellular protease activities of AP57 and ?P70 were decreased by above 50%.2.Characterization of the nitrogen regulation factor Arel that modulates nitrogen metabolite repression and extracellular protease and cellulase expression in T.reeseiUsing the amino acid sequences of A.nidulans AREA and N.crassa Nit2 as the query,the nitrogen regulator Are1 was identified in T.reesei.In order to functionally characterized the Are1 in T.reesei,we constructed the are 1 deletion strain using the homologous recombination,the ?are1 mutant was reduced in protease and cellulase activity and the expression levels of the major cellulase genes cbhl,cbh2,eg1,eg2 and bgll were significantly decreased.In filamentous fungi,the predicted DNA binding site for Are1 is 5'-HGATAR-3'(where H represents A,T,and C and R represents A or G).By predicting the potential binding site of Arel in the promoter region of the cellulase gene,we have found that the promoter of cellulase gene contained the Are1 binding sites.Therefore,Are1 may directly regulate the expression of cellulase.Similar to function of the other filamentous fungi Arel homologues,T.reesei arel deletion strain could not utilize the nitrates as nitrogen source.However,arel deletion strain unable to utilize ammonium as sole nitrogen source.Analysis of the RNA-seq data,we found that arel positively regulates the expression of related genes in the ammonium metabolic pathway of T.reesei.Ammonium permeases mepl and mep2,glutamate dehydrogenase gdhl,and glutamine synthetase gsl expression were significantly decreased in ?are1.We overexpressed mep1,mep2,gdh1,and gs1and found that overexpression of mep1,mep2,and gdh1 can restore the ability of ammonium utilization,while overexpression of gsl unable restore the the ability of ammonium utilization.In summary,we identified a nitrogen regulator Arel in T.reesei and found that Arel regulate the expesssion of genes associated with ammonium metabolism,anmio acid synthesis,protease gene and cellulase gene.It is also demonstrates that Arel plays an important role in the regulation of nitrogen sources and development in filamentous fungi.3.p53-like regulation factor Vibl positively regulates cellulase and protease expression in T.reesei.The regulation of extracellular proteases of A.nidulans was not only regulated by AreA,but also regulated by specific transcription factor XprG.Therefore,we studied the function of Vibl,a homolog of XprG,in T.reesei.Using gene deletion and overexpression,we have foud the extracellular protease activity of the vibl deletion strain was significantly reduced.We also found that ?vibl unable to production cellulase and could not growth on the cellulose.Overexpression of vibl resulted in significantly increased in cellulase activities.The activities of FPA,EG,CBH,BGL and protein concentration increased by 70%,80%,77%,110%and 40%,respectively.We further analyzed the expression levels of major transcription factors in?vibl mutant and found that the expression level of the key cellulase transcriptional activator xyrl was significantly reduced.However,overexpression of xyrl failed to restore the cellulase secretory capacity in Avibl mutant.This data suggested that Vibl may not regulate cellulase expression by directly regulating xyrl expression.Transcriptome data revealed that the expression level of the cellobiose transporter Crtl was significantly reduced in Avibl mutant,and the lack of Crtl in T.reesei resulted in the inability of the strain to produce cellulase activity,and the expression of xyrl was also significantly reduced.It may be suggested that Vib1 may regulate the expression of crtl and then regulate the expression of cellulase.4.Characterization and strain improvement of a hypercellulolytic variant,Trichoderma reesei SN1,by genetic engineering for optimized cellulase production in biomass conversion improvementThe filamentous fungus T.reesei is a widely used strain for cellulolytic enzyme production.A hypercellulolytic T.reesei variant SN1 was identified in this study and foundto be different from the well-known cellulase producers QM9414 and RUT-C30.The cellulose-degrading enzymes of T.reesei SN1 show higher endoglucanase(EG)activity but lower ?-glucosidase(BGL)activity than those of the others.A uracil auxotroph strain,SP4,was constructed by pyr4 deletion in SN1 to improve transformation efficiency.The BGL 1-encoding gene bgl1 under the control of a modified cbhl promoter was overexpressed in SP4.A transformant,SPB2,with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30.0%increase in filter paper activity.Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65.0%higher than that of SP4.These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion.