Regulation Of Cellulase Induction And Optimization Of Recombinant Protein Expression In Trichoderma Reesei

Filamentous fungus Trichoderma reesei can produce large amounts of cellulases when cellulosic substrate is used as carbon source.Deciphering the mechanism of cellulase induction would greatly benefits for the thorough understanding of the cellulase regulation networks,as well as for the hyper-cellulase production.Till now,the majorities of studies mainly focus on the discovery of novel transcription factors which regulates cellulase expression at transcription level.However,during the cellulase induction,several regulators including sugar transporters,as well as secretion facilitating factors are involved in cellulase expression.The detailed functions of these regulators in cellulase expression still need further study.Moreover,T.reesei has been considered as a‘Generally recognized as safe’strain,and has been used for producing over 20 commercial recombinant proteins.In most cases,the recombinant protein production in T.reesei was conducted using its own cellulase regulating networks.Based on this,it is great value to deepen the mechanism of cellulase expression and to uncover the key regulators,which benefit for rational engineering for recombinant protein expression using its cellulase regulating networks.Our study mainly focused on two areas:the discovery of novel regulator for cellulase expression,and construction of recombinant protein expression platform based on cellulase regulating networks,and the results were listed as follows.1.Uncovering the key elements for vacuolar protein sorting and its regulatory mechanism on cellulase expression.Screening and functionally characterizing eleven putative vacuolar protein sorting related factors in T.reesei.We found that deletion of vps10,vps13 and vps21 improved cellulase production（Filter paper activity,FPase）in T.reesei by 1.28-,2.45-,2.11-fold than that of the parent strain.Furthermore,we also tracked the vacuolar transport of vacuolar peptidases Cpy and Pep4,and found that the vacuolar trafficking towards vacuoles was partially impaired in three knockout mutants.Mis-secretion of Cpy and Pep4extracellularly was also observed.Moreover,alleviated autophagy was found when vps13 was disrupted.These results indicated that alleviated transport and degradation towards vacuole inΔvps10,Δvps13 andΔvps21 might improve cellulase production.Further studies indicated that the expression of cellulase genes inΔvps13 andΔvps21 was dramatically increased in the late induction phase compared to the parent.Further transcriptome analysis revealed that the sugar transporters were differentially expressed in late phase,which provides the potential engagement of sugar transporter in high-level cellulase expression in T.reesei.2.Identification of a cellulose response transporter like protein Crt2 and the deciphering of its involvement in cellulase induction.To investigate the effect of nine putative sugar transporters on cellulase expression,we found that the constitutive expression of a cellulose response transporter like protein Crt2（Tre77517）improves cellulase induction.Functional studies indicate that Crt2 is not a transporter of cellobiose,lactose or glucose in yeast system,and it also does not affect sugar utilization in T.reesei.Further studies revealed that the function of Crt2 is similar to that of the major cellobiose transporter Crt1 in cellulase induction.The expression of crt2 is activated by cellulosic substrates.Then,Crt2 acts as a signal transceptor through transducing the cellulose-inducing signal to the downstream transcription factor Xyr1,which then activates the cellulase gene expression.Besides,the Crt1,Crt2,and Xyr1 forms a regulation network during cellulase induction,suggesting that the cellulose response transporter like protein Crt2 could participate in the cellulase induction with Crt1collaboratively.3.Design and optimization of the recombinant protein expression platform based on the cellulase transcriptional regulation networks.We constructed a recombinant protein platform using the cellulose-inducing promoter Pcbh1.The binding module of transcription activator Xyr1 and repressor Ace1 was rearranged,then seven recognition sites of the inverse repeat of Xyr1 were added on Pcbh1 promoter,and the recognition sites of Cre1 is deleted.This resulted in a 4-fold improvement in transcription level and 2-fold improvement in recombinant protein production in a Xyr1-overexpressing strain（OExyr1/7IRGFP）,compared to the parent strain C30/GFP.Improved recombinant lipase Cal B expression is achieved using the engineered promoter Pcbh7IR,however,activated unfolded protein response（UPR）as well as a feedback mechanism called repression under secretion stress（RESS）is still observed.Further study through adding the binding sites of UPR related transcription factor Hac1 only slightly improves the Cal B expression on strain C30/Cal B which harbored the native Pcbh1 promoter,but failed to reach a higher Cal B production based on the engineered promoter.4.Effect of engineering vacuolar protein sorting and cellulose inducing signal on recombinant protein expression,and its fed-batch culture optimization.The deletion of vps10,vps13 and vps21 in Cal B expressing strain cannot further improve the Cal B production,but lead to impaired mycelia accumulation in Avicel inducing medium,which hinder the Cal B production.The complement of major cellulase Cbh1 through a moderate promoter Pegl1 in vps10-deleting strain improves the Cal B production by 117.6%.Moreover,Crt2 overexpression only activate the transcript of cal B to 2.66-fold in the early stage of cellulose induction,but could not improve the Cal B production in the culture.Finally,the carbon sources,nitrogen sources as well as the feed modes were optimized in a 3-L bioreactor,reaching a recombinant Cal B production level up to 1.80 g·L^-1,and the recombinant xylanase Xyn2 production level up to 2.33 g·L^-1.These results suggested that the recombinant protein expressing platform built in this study is potentially applied in industry fermentation.