Study On Transcript-level Regulation Of Cellobiohydrolase Cel7A And Its Enzymatic Catalysis In Trichoderma Reesei

The filamentous ascomycete Trichoderma reesei encodes a range of cellulase,and shows high production of cellulase widely used in biofuel production,paper making and feed processing,etc.,and then has become one of the most studied microorganisms.However,carbon catabolite repression(CCR)in T.reesei was triggered in the presence of glucose,and then the expression of cellulase was repressed.Therefore,how to alleviate CCR is an important science question for developing the technology for producing the cellulolytic enzymes.Meanwhile,the nonproductive adsorption of cellulase onto lignin significantly inhibited the enzymatic hydrolysis of lignocellulose.Many scientists focused on mechanism of nonproductive adsorption of lignin and how to increase the partition coefficient of enzymes between Avicel and lignin for improving the enzymatic hydrolysis efficiency of lignocellulose and reduce the cost of enzyme preparation.The contents in this thesis were described as follows:1.Constructing the cellulase self-induction system for improving Cel7A productionIn T.reesei,GH family 1 β-glucosidase-cel1a or cel1b play an important role in morphology of mycelium and cellulase production.There is no obvious change of sporogenesis and cellulase synthesis between parent strain and mutant without cell a.The disruption of cel1b in T.reesei results in obvious defects in sporogenesis and has a great influence on cellulase production.Furthermore,the sporogenesis and cellulase production in mutant with the simultaneous deletion of cella&cel1b were inhibited,compared to those in the single deletion mutants.We replaced the Cel1b with its variant-Cel1bI174S/I177S in T.reesei and generated M5M8.We found that there is no change of morphology of mycelium between parent strain and M5-M8(M5:ΔCellb::CellBI174S/I177S,M6:ΔCellb::OE Cel1BI174SI177S,M7:ΔCel1a&ΔCel1b::Cel1BI174S/I177S and M8:ΔCel1a&ΔCel1b::OE Cel1BI174S/I177S).Cellulase production in M5-M8 were greatly improved,compared with that of parent strain using the mixture of cellulose and glucose as carbon source.We proved that the function of cel1a for sporogenesis and cellulase synthesis significantly differed from those of cel1b.Our findings revealed that CEL1B plays a positive role on transcriptional regulation of cellulolytic enzyme in T.reesei and disruption of CEL IB has a somewhat negative effect on sporogenesis and cellulase production.Meanwhile,its modification significantly improved the transcriptional levels and synthesis amount of cellulolytic enzyme when glucose existed in the media.2.Modifying transcription factor Cre1 for improving Cel7A expressionCCR,which is mainly mediated by Crel and triggered by glucose,leads to a decrease in cellulase production in T.reesei.Many studies have focused on modifying Cre1 for alleviating CCR.Based on the homologous alignment of CreA from wild-type Penicillium oxalicum 1142(Po-0)and cellulase hyperproducer JUA10-1(Po-1),we constructed a C-terminus substitution strain-Po-2,and the transcriptional levels of cellulase in strain Po-2 was repressed,compared to that of parent strain.Results revealed that the C-termina1 domain of CreAPo-1 plays an important role in alleviating CCR.Furthermore,we replaced the C-terminus of Cre1 with that of CreApPo-1 in T.reesei(Tr-0)and generated Tr-1.As a control,the C-terminus of Crel was truncated and Tr-2 was generated.The transcriptional profiles of these transformants revealed that the C-terminal chimera greatly improves cellulase transcription in the presence of glucose and thus upregulates cellulase in the presence of glucose and weakens CCR.Furthermore,we found that there is a conserved region at the C-terminus of Crel/CreA in several cellulase-producing fungi that contains up to three continuous S/T phosphorylation sites.Here,S387,S388,T389,and T390 at the C-terminus of Cre1 in Tr-0 were mutated to valine for mimicking an unphosphorylated state,thereby generating the transformants Tr_Cre1S387V,Tr_Cre1S388V,Tr_Cre1T389V,and Tr_Cre1T390V,respectively.Transcription of cel7a in Tr_Cre1S388V was markedly higher than that of the parent strain when grown in glucose-containing media.Under these conditions,both filter paperase(FPase)and p-nitrophenyl-β-D-cellobioside(pNPCase)activities,as well as soluble proteins from Tr_Cre1S388V were significantly increased by up to 2-to 3-fold compared with that of other transformants and the parent strain.Additionally,it was found that S388 maybe be phosphorylated by cyclic adenosine monophosphate(cAMP)-dependent protein kinase A(PKA),a conserved key factor of a nutrient-sensing pathway that acts in parallel to the MAP kinase pathway and then regulate CCR.Taken together,we proved that there is a direct relationship between the C-terminal chimera of Crel and CCR,and developed a precision engineering strategy based on the modification of phosphorylation sites of Cre1 transcription factor to enhance the production of cellulase in fungal species under CCR.3.Enhancing lignocellulosic degradation efficiency of Cel7A with the addition of chemicalsThe nonproductive adsorption of cellulase onto lignin significantly inhibited the enzymatic hydrolysis of lignocellulosic biomass.In this study,we constructed a rapid fluorescence detection system(RFD),and using this system,we demonstrated that the addition of cationic additives-DTAB or polyDADMAC greatly increased the partition coefficients of cellulose/lignin,reduced nonproductive adsorption,and enhanced the hydrolysis efficiency of lignocellulose compared to those of Tweens or PEGs.Moreover,the addition of polyDADMAC and DTAB increased the glucose yield released from the mixture of Avicel and AICS-lignin(MCL)by 16.9%and 20.6%,respectively.Interestingly,polyDADMAC or DTAB treatment performed more effectively for enzymatic hydrolysis of pretreated lignocellulosic biomass,compared with MCL.We confirmed that reduced hydrophobicity and increased zeta potential of lignin co-contribute to the dampening nonproductive adsorption of lignin.In particular,the zeta potential values of lignin and the partition coefficients of Avicel/lignin with the addition of additives showed a good correlation,suggesting that electrostatic force also plays a crucial role in the adsorbing of cellulase on lignin.This work will be conducive to decreasing the nonproductive binding of cellulase onto lignin and enhancing cellulose conversion,and providing a theoretical basis for the rational design of CBM to alleviate nonproductive adsorption of lignin onto cellulase.