Study On Mechanism Of New Transcription Factors Of Trichoderma Reesei

Trichoderma.reesei is commonly utilized in industry to produce cellulase,an enzyme that degrades lignocellulosic biomass for the production of bioethanol and bio-based products.T.reesei is capable of rapidly initiating the biosynthesis of cellulase in the presence of cellulose,which has made it useful as a model fungus for studying gene expression in eukaryotes.Cellulase gene expression is controlled through multiple transcription factors at the transcriptional level.However,the molecular mechanisms by which transcription is controlled remain unclear.In this study,we screened out two novel transcription factors,the positive transcription factor ACE4 and the negative transcription factor RCE2.This paper expands from the following two aspects:1)We identified by comparative genomic screening a novel transcriptional activator ACE4(Activator of cellulase expression 4)that positively regulates cellulase gene expression in T.reesei.Disruption of the ace4 gene significantly decreased expression of four main cellulase genes,and the essential cellulase transcription factor encoding gene ace3.Overexpression of ace4 increased cellulase production by approximately 21.9%compared to that in the parental strain.Further investigations using electrophoretic mobility shift assays,DNase I footprinting assays,and chromatin immunoprecipitation assays indicated that ACE4 directly binds to the promoter of cellulase genes by recognizing the two adjacent 5 '-GGCC-3' sequences.Additionally,ACE4 directly binds to the promoter of ace3 and,in turn,regulates the expression of ACE3 to facilitate cellulase production.2)We isolated a novel protein,RCE2(Repressor of Cellulase gene Expression 2),using a pull-down assay and mass spectrometry analysis,from a partial carbon catabolite de-repression mutant T.reesei Rut-C30 cultured under glucose-repressing conditions.Deletion and overexpression of RCE2 in T.reesei wild-type QM6a revealed that RCE2 acts as a repressor of cellulase gene expression.DNase I footprinting assays,electrophoretic mobility shift assays(EMSA),and Chromatin immunoprecipitation assays(ChIP)assays revealed that RCE2 can be located in the nucleus and can bind to the consensus sequences 5'-(T/A)NNNNCCG-3' and 5'-CGGNNNN(T/A)-3' in the promoters of cellulase-related genes to repress their transcription.Additionally,RCE2 antagonized ACE3 binding to the cbh1 promoter and repressed the transcription of xyr1 to down regulate cellulase gene expression.Moreover,RCE2 was not involved in Crel-mediated carbon catabolite repression.Collectively,these results demonstrate an important role for ACE4 and RCE2 in regulating cellulase gene expression,which will contribute to understanding the mechanism underlying cellulase.These results also provide new methods that can be used for increasing cellulase production in T.reesei.