Study On The Mechanism Of The Transcriptional Activator Xyr1 In The Induced Cellulase Gene Expression In Trichoderma Reesei

The ?-(1,4)-linked glucose polymer cellulose is the product of photosynthesis of plants and algae by using solar energy and carbon dioxide and is one of the most abundant carbohydrates on earth.The degradation of this polysaccharides thus constitutes a major transformation step in the biological carbon cycle in nature.Among the numerous cellulose degrading microbes in nature,the filamenouts ascomycete Trichoderma reesei is a typical saprobic fungus capable of efficiently degrading cellulose to glucose or cellulooligosaccharides.Trichoderma reesei is able to express and secrete large amount of cellulases when exposed to cellulose,thus making it an important industrial cellulase producing strain for the production of biofuels.After decades of mutagenesis study and breeding,several hyperproducer strains have been obtained and the cellulase poductivity of this fungi has greatly improved.But the still high cost of cellulase in transforming cellulose materials is still a bottleneck for the successful development and application of biofuels due to the low poductivity and hydrolytic efficiency of cellulases.Despite the tremendous progress that has been made in the extensive characterization of cellulase in T.reesei,the molecular mechanism underlying the regulation of cellulase production in this fungi is still insufficiently understood.At present,several transcription factors that are involved in regulating cellulase gene expression have been identified in T.reesei,including four positive regulators(Xyr1,Ace2,Ace3 and the HAP2/3/5 complex)and two repressors(Ace1 and Cr1l).Xyr1(Xylanase regulator 1)has been proved to be the predominant activator of cellulase and hemicellulase gene expression since its deletion eliminates cellulase induction by all inducers and also impairs the induction of different hemicellulase genes involved in xylan degradation.Although its role in(hemi)cellulase gene regulation has been characterized,the mechanism by which Xyr1 triggers the induced expression of those genes is still unclear and awaits further investigation.This research focus on the transactional mechanism of Xyr1,including the identificationof its potential interacting partners and exploring the function of the key subunits of two candidate co-activator complex of Xyr1.The main results of this study are as follows:1.The mating type locus protein MATal was identified as a novel interacting partner of Xyr1 in yeast two-hybrid screen.Further investigation revealed that MATal regulated cellulase gene expression in response to light and was recruited to cellulase gene promoter by Xyr1.In order to identify any interacting protein of Xyr1,a yeast two-hybrid(Y2H)based cDNA library from T.reesei induced on cellulose was constructed.Several positive clones were obtained in the Y2H screen,one of which was identified to be the mating type locus protein MATa1.Transcriptional assay revealed that natalgene expression was up-regulated under both cellulose inducing conditions and in light.The encoding gene of this protein was then knocked-out in T.reeseiTU-6 strain.Whereas deletion of matalled to decreased expression of cellulases in light,there was hardly any difference regarding cellulase gene expression between the ?matal strain and the parental strain in constant darkness.These results thus indicated that MATa1 was involved in cellulase gene regulation in response to light.MATa1 constitutively localized in nucleus and showed co-localization with Xyr1.Further ChIP assay revealed that MATa1 was recruited to the cbh1 promoter and this recruitment was dependent on the induction signal.In contrast,MATa1 occupation on hppl and hpr2 promoters was constitutive regardless of what carbon source was used.In the xyrl deleted strain,MATal recruitment to the cbhl promoter was almost abolished even under inducing conditions,indicating that Xyr1 was critical for its recruitment.Taken together,our study revealed that MATa1 was a novel interacting partner of Xyr1 and it acts coordinately with Xyrl to regulate cellulase gene expression in response to light.2.Turn off/down the expression of histone acetylation transferase complex subunits Gcn5,Ada2 and Ada3 abolished or dramatically decreased the induced expression of cellulases in T.reesei,indicating that this complex plays an important role in cellulase gene expression.Previously we had shown that GCN5 was essential for the induced expression of cellulase genes in T.reesei.In this study we developed a promoter replacement system that allowed the one-step substitution of the target gene promoter for the copper responsive promoter Ptcu1.This replacement put the expression of the target gene under the control of copper.We then showed that the controlled expression of the histone acetyltransferase genegcn5 achieved by this strategy displayed the same phenotype in the presence of copper as the gcn5 deletion strain described in our previous work.We then characterized the two other associated subunits of Gcn5,Ada2 and Ada3,which have been shown to play important roles in modulating the activity of GCN5.The promoters of ada2 and ada3 gene were replaced by Ptcu1 to obtain two recombinant strains Ptcu1-ada2/ada3 and their phenotypes were assayed by adding copper.We found that turning off the expression of ada2 almost abolished the induced expression of cellulased indicating that Ada2 plays a vital role in cellulase gene expression in T.reesei.This strain also showed a growth defect and more sensitive to several stress conditions when copper was added.Similarly,turn-off of the expression of ada3 by copper also led to a dramatically decreased expression of cellulase genes.Taken together,these results indicated that histone acetylation transferase complex plays an important role in the induced expression of cellulases in T.reesei.3.Xyrl targeted recruitment of Gcn5/Ada2/Ada3 complex regulates cellulase gene expression.To further investigate the relationship between Xyrl and the histone acetylation transferase complex,ChIP assay was conducted to detect the occurrence of H3K9 and H3K14 acetylation at cellulase gene promoter in the xyrl deletion strain ?xyr1.We found that both H3K9 and H3K14 acetylations were almost abolished in the Axyrl strain even under inducing conditions.Further analysis revealed that Xyr1 interacted with GCN5 and Ada3 in Y2H assay,thus implying that GCN5 cooperates with Xyrl to regulate cellulase gene expression.To study the relationship between Xyrl and GCN5 in vivo,a ProA-Gcn5 recombinant strain was constructed and ChIP assay with this strain revealed that GCN5 was recruited to cellulase gene promoters under cellulose inducting conditions.Further analysis revealed that the GCN5recruitment to cellulase gene promoters could bypass by the inducing signal when xyr1 was overexpressed.These results thus indicated that GCN5 was recruited to cellulase gene promoters in a Xyr1 dependent manner and this recruitment was mediated by their direct interactions.However,turning off the expression of gcn5 in the OExyr1_Ptcul-gcn5 strain did not affect the expression of cellulases,which implying that overexpression of Xyr1 could bypass the function of GCN5.In summary,we found that Xyrl targeted recruitment of GCN5 to cellulase gene promoters is involved in the regulation of cellulase gene expression and the function of GCN5 partially controls the expression of xyrl.4.Gal11 of the Mediator complex was differentially involved in the induced expression of cellulase in T.reesei.Deletion of gal11/med15 resulted in a dramatically decreased expression of cellobiohydrolases and endoglucanases while having little impact on ?-glucosidaseexpression.Mediator plays a central role in the regulation of RNA Pol ? transcribed genes in eukaryotes.In Scharomyces cerecisiae,many transcription activators including Gal4 and Gcn4 et.Al,act to recruit the Mediator complex through its tail module subunit Galll/Medl5 to initiate target gene expression.To investigate whether Gal11 participated in Xyr1 mediated activation of cellulase genes,Gal11 was identified by using bioinformatics method and its encoding gene was knocked out in T.reesei.We found that deletion of Gal11 did not affect growth on solid plate and liquid medium with several carbon souces,but abolished conidiation and yellow pigment formation,indicating that Gal11 playd a vital role in asexual reproduction and secondary metabolism in T.reesei.Enzymatic and transcriptional assays revealed that the expression level of cellobiohydrolases and endoglucanases was dramatically decreased in the ?gal11 strain when cultivated on cellulose medium,while the expression of ?-glucosidase was unaffected.Deletion of gal11 also resulted in a significantly reduced expression level of xyr1.Taken together,those results indicated that Gal11 differentially regulates cellulase gene expression in T.reesei and it was required for the foll induced expression of cellobiohydrolases and endoglucanases.5.Gal11 participates in regulating cellulse gene expression by compromising RNA Pol ? recruitment and the stability to Xyrl bound to cellulase gene promoter.ChIP assay revealed that the occupation of RNA Pol ? on the core promoter of cellobiohydrolase and endoglucanase genes was dramatically decreased in the ?gal11 strain while these was hardly any difference for its occupancy at the ?-glucosidase gene promoter between the ?gal11 and the parental strains,which was in accordance with the phenotypes of this strain.Further analysis revealed that,although the expression level of xyrl was significantly reduced in the ?gal11 strain,its occupancy at cellulase gene promoters were much higher that the parental strain,indicating that deletion of gal11 may result in an increased stability of Xyr1.A recombinant strain OExyr1_?gal11 that simultaneously overexpressed xyr1 and knocked out gal11 did not restore the expression level of cellulases on cellulose.The induction-independent expression of cellulases on glucose mediated by xyr1 overexpression was also almost abolished in this strain.Furthermore,interaction between Xyr1 and Gal11 was detected in the Y2H assay.Taken together,Gal11 may directly participate in transcription regulation by mediating the recruitment of RNA Pol ? and affecting the stability of Xyr1.6.Overexpression of Xyr1 resulted in an induction-independent production of cellulases.RNA-seq assay revealed that overexpression of Xyr1 was sufficient to activate most(hemi)cellulase gene expression under non-inducing conditions.Previously we found that overexpression of xyr1 by tcul promoter resulted in induction-independent expression of cellulases.In this study,a recombinant strain that overexpressed Xyr1 in the endogenous pyr4 locus was constructed(OExyr1).Phenotypic analysis revealed that both cellulase production rate and total enzymatic activity of the OExyr1 strain cultivated in glucose medium were much higher than those of QM9414 in cellulose.Further analysis revealed that,compared to the higher level of cellobiohydrolases and endoglucanases in the OExyr1 strain,the?-glucosidase activities were significantly lower that the parental strain,suggesting that overexpression ofXyr1 did not effectively active the expression of ?-glucosidase genes.ChIP assay revealed that overexpression Xyr1 resulted in dramatically decreased occupation of histones on cellulase gene promoters and this effect could be restored by turning off the expression of Xyr1,indicating that regulating the chromatin structure of cellulase gene promoter was an important event during the Xyr1-initiated transcription of those genes.RNA-Seq was applied to further determine the transcriptional pattern of the whole genome when overexpressing Xyr1,The results showed that overexpression of Xyr1 caused the up-regulation of 589 genes and the down-regulation of 3637 genes on glucose compared to QM9414.Under cellulose inducing condition,897 and 1012 genes showed up-regulation and down-regulation in QM9414,respectively.Cluster analysis revealed that there were 334 overlapping genes between those two up-regulated gene groups,comprising almost all the cellulase and hemicellulase genes.Most of the cellulase gene transcriptional activators and MFS transporters showed induced up-regulation in QM9414,while this was not the case in the OExyr1 strain,suggesting that overexpression of Xyr1 alone was sufficient to activate the full expression of cellulase genes.Several important genes involved in protein folding and secretion showed significant up-regulation in the OExyr1 strain,indicating that overexpressed Xyr1 also activated the ER associated pathways to facilitate cellulase secretion.Functional cluster analysis of the differentially expressed genes revealed that numerous primary metabolism pathway genes showed down-regulation in the OExyr1 strain,including that glycolysis,TCA cycle,amino acid biosynthesis and oxidative phosphorylation and so on.Taken together,we proposed that overexpression of xyr1 led to the diversion of metabolic fluxes to the efficient syntesis and secretion of cellulases.