Investigation Of The Role Of CCR In Cellulase Gene Expression In Trichoderma Reesei

With the increasing environmental pollution caused by burning of fossil fuels,as well as the depletion of the non-renewable resources,an environmentally clean and renewable energy is urgently needed.Biofuels derived from lignocellulose,which is widely found and rich in nature,could partially replace our demands for fossil fuels,and reduce environmental pollution.Cellulase could efficiently degrade lignocellulosic biomass to soluble sugars,which can further be fermented to biofuels such as ethanol.As the main cellulase producers,the filamentous fungi are widely used in industrial production of various cellulases.Among others,Trichoderma reesei has been studied in depth for its ability in producing large amounts of cellulase efficiently.In the presence of insoluble cellulose,hemicellulose and plant biomass,T.reesei could secrete celllulase efficiently.At the same time,the expression of cellulase is also regulated by carbon catabolite repression(CCR),which ensures the strain to use glucose in priority.The induction mechanism of cellulase expression has been studied extensively,however,the mechanism of carbon catabolite repression in regulating cellulase expression received less attention.Cre1 is the key transcriptional factor mediating carbon catabolite repression in T.reesei,whose deletion leads to higher cellulase expression in both inducing and repressing conditions.But there are still many questions that need to be answered,such as how T.reesei senses glucose,how the inhibiting signals are transmitted,how Cre1 regulates transcription of cellulase gene at the molecular level and so on.In this study,we focused on the following five areas:1.Constructed crel gene knockout and overexpression strains to investigated its biological role.Cre1 is a C2H2 zinc finger protein which plays a key role in carbon catabolite repression in T.reesei.We constructed crel gene knockout strain(Acre1)and crel overexpression strain(Ptcu-crel)by means of homologous recombination and verified them by anchored PCR.Phenotypic analysis showed that Acrel grows much weaker than that of wild type on various carbon sources in the plates,forms irregular colony edges and produces less spores.Microscopy results showed that no significant difference could be detected in mycelia morphology between the Acrel strain and wild type strain.Ptcu-cre1grows similar to that of the WT strain,but exhibits no cellulase formation under inducing conditions.2.Analyzed the role of Crel in turning on and off the transcription of cellulase gene.Previous study showed that crel inhibited cellulase expression both at basal level and induction level.We further analyzed the impact of Crel in turning on and off cellulase gene transcription.Our results showed that cellulase gene transcription was delayed in Acrel.In addition,the efficiency of transcription turning off was also affected by the absence of Cre1.In conclusion,we found that Cre1 plays important role in turning on and off the transcription of cellulase gene.3.Studied the intracellular localization of Crel under different carbon sources and investigated the role of in vivo binding of Crel to promoters in regulating cellulase expression.In order to probe into the molecular mechanism of Crel in regulating cellulase expression,we investigated its intracellular localization by fluorescent microscopy.We found that Crel stayed in the nucleus both under inducing and inhibiting conditions.To further investigate the role of Cre1 binding to cellulase gene promoters,we constructed a mutant with mutations of three main Cre1 binding sites in cbhl promoter called Pcbh1 MT.Our results showed that,in inconsistent with data obtained from Acrel,cellulase expression was not delayed in Pcbh1 MT,but was advanced.In conclusion we found that in vivo binding and nucleocytoplasmic shuttling of Crel did not play an important role in activating cellulase expression.4.Identified the cofactor protein Trtc1 and analyzed its role in regulating cellulase expression.In Saccharomyces cerevisiae,Miglp(homologous protein of Crel)mediated suppression mainly through corepressor Tup1p.We identified the Trtc1(Tup 1-related transcriptional cofactor 1)protein in T.reesei by sequence alignment and studied its function.We replaced the native trtcl promoter by a copper responsive promoter and regulated the transcription of trtcl by adding copper.We found that trtcl deletion lead to severe defect in cellulase expression,while its overexpression increased the expression of cellulase.