Study On The Regulatory Mechanism Of Xylanase Expression In Trichoderma Reesei

Lignocellulose is the most abundant renewable resource on the earth.It mainly consists of cellulose(～50%),hemicellulose(～25%)and lignin(～25%).Among others,the main component of hemicellulose is xylan with complex structure,which is often highly branched and thought to exist between lignin and cellulose.The effective degradation of xylan plays an indispensable role in increasing the accessibility of cellulase to cellulose and thus improving the overall degradation efficiency of lignocellulose.Among lignocellulose-degrading microorganisms,the filamentous fungus Trichoderma reesei has been the main industrial strain for producing cellulases and hemicellulases that is widely used in energy,food,textiles and paper making.In recent years,extensive efforts have been made to elucidate the mechanism of cellulase induced biosynthesis in T.reesei,which also forms the basis for rational strain transformation to achieve improvement of enzymatic performance and increase in yield.Compared with the research on deciphering the regulatory network controlling cellulase synthesis including a variety of positive and negative regulatory factors,the research on the regulation mechanism of hemicellulase synthesis mainly represented by xylanases in T.reesei is lagging behind,and only a few studies have been reported.However,there are a variety of xylanase genes in T.reesei and their expression regulation is also an extremely complex process.The induced biosynthesis of hemicellulases also initiates from sensing environmental signals to eventually resulting in xylanase gene expression,wherein a variety of different protein factors are believed to participate in.Therefore,the identification of the regulatory factors of xylanase gene expression in T.reesei and the analysis of its regulatory mechanism can not only provide a deeper understanding of the regulatory mechanism of xylanase gene expression at the molecular level,but also deepen and enrich our understanding of the overall regulatory network that help facilitate the efficient coordinated degradation of lignocellulose by T.reesei.Therefore,this study has both important theoretical significance and potential application value.Based on the above propositions,this paper carried out a series of studies on the expression regulation of xylanase genes in T.reesei,and the main research results are as follows:1.XYNⅡ was identified as the main contributor to the overall extracellular xylanase activities of Trichoderma reesei,and the deletion of xyn2 promoted the expression of xynl.In this study,Δxynl and Δxyn2 strains were constructed and antibodies against XYNⅠ and XYNⅡ were prepared,respectively.The extracellular xylanase activities of Δxym1 and Δxyn2 mutant strains was then detected.It was found that each antibody could specifically recognize the corresponding xylanase without any cross reactivity.Further analysis of extracellular xylanase activities,transcription level of xylanase genes and expression kinetics of Δxyn1 andΔxyn2 strains revealed that the deletion of xyn1 did not affect the expression and secretion of XYNⅡ or the total activities of xylanase in extracellular fermentation broth.In contrast,the protein expression and secretion of XYNⅠ increased by two times with deletion of xyn2 although the overall xylanase activities in extracellular fermentation broth was found at a much lower level.These results indicated that XYNⅡ was the main contributor to xylanase activities in the extracellular fermentation broth of T.reesei,and the deletion of xyn2 promoted the expression of xyn1.2.Isolation and identification of galacturonic acid transporter GAT1 as a new regulator of Trichoderma reesei xylanase gene expressionTranscriptomic analysis of the wild-type strain QM9414 under xylan and glucose carbon source showed that the expression of the galacturonic acid/glucuronic acid transporter GAT1 was significantly upregulated under induced carbon source.By analyzing the phenotypes of gatl knockout and overexpressed strains under xylan,it was found that xyn1 was the main regulatory target of GAT1 with its expression being significantly increased in the absence of GAT1.In addition,the pectin hydrolysate galacturonic acid was found to inhibit the expression of pectinase genes pecl,pec2 and xylanase gene xynl and this inhibition was relieved upon gatl deletion.However,addition of glucuronic acid did not inhibit the activities of xylanase in QM9414 strain under the conditions of xylan,indicating that the effect of GAT1 on xylanase expression was probably independent of its glucuronic acid transport activities.Transcriptomic data of Agatl and QM9414 induced by xylan were analyzed to identify candidate genes that may be regulated by GAT1.Among them,the expression of a transcription factor Tr82512 in Δgat1 was most up-regulated.The analysis of xylanase activities of the Tr82512 overexpressed strain showed that Tr82512 overexpression increased extracellular xylanase activities,indicating that the deletion of gatl may lead to the up-regulation of Tr82512 expression which promote the expression of xylanase I.In summary,GAT1 specifically targets and inhibits the expression of xylanase gene xyn1.GAT1 does so probably by regulating the transcription level of the transcription factor gene Tr82512.3.A novel transcriptional repressor,XTR1,from Trichoderma reesei was identified and demonstrated to be involved in the regulation of xylanase gene xynl by directly binding to its promoterA transcription factor XTR1 with a Cys2His2 DNA-binding domain was identified from the cDNA library of T.reesei using the promoter of xylanase gene(Pxyn1)as a "bait" in yeast one-hybrid screening.Subcellular localization analysis revealed that XTR1 was mainly located in the nucleus.The deletion and complementation phenotypes of xtr1 were analyzed,and it was found that the xyn1 promoter was the main regulatory target of XTR1 to repress the expression of xylanase gene.The interaction between XTR1 and xyn1 promoter was verified by electrophoretic mobility shift assays(EMSA)in vitro,and the precise binding region of the promoter by XTR1 was located to 105 bp(-321～-217).Finally,the precise binding site of XTR1 on the xynl promoter was analyzed by DNase I footprinting assay.In conclusion,the transcription factor XTR1 screened by yeast one-hybrid screening participates in the regulation of xylanase gene expression by binding to the promoter of xyn1.4.Construction of a Trichoderma reesei strain with high-level production of cellulases and xylanases under repressive carbon conditionsXYR1 is the key transcriptional activator that mediates almost all cellulase and hemicellulase gene expression in T.reesei.Overexpression of XYR1(OExyr1)resulted in constitutionally high levels of cellulase gene expression in the presence of glucose,but xylanase gene was not activated as well.In order to futher improve the xylanase activities of the OExyr1 strain,we constructed OExyr1-Δsxlr by knocking out the xylanase repressor SxlR,which has been shown to regulate xyn1,xyn2 and xyn5.By analyzing the enzyme activities of the OExyr1Asxlr strain,it was found that the deletion of sxlr significantly increased the xylanase activities and β-glucosidase activities under Avicel or glucose conditions.Subsequently,the chromatin immunoprecipitation(ChIP)assay showed that the overexpressed XYR1 in OExyr1-Asxlr strain significantly increased its occupancy at the xyn1 promoter compared with the OExyrl strain,thereby increasing the expression of xyn1 and increasing the extracellular xylanase activities.Saccharification assay with the extracellular enzyme cocktail of OExyrl-Δsxlr showed that the fermentation broth of OExyr1-Δsxlr had better saccharification ability towards wheat bran and corn husk than that of the OExyr1 strain obtained under glucose conditions.In conclusion,through the simultaneous overexpression of XYR1 and deletion of SxlR,a strain with high yield of cellulase and xylanase produced under the condition of soluble repressing carbon source was obtained.The resultant crude enzymes displayed improved saccharification efficiency towards natural substrates and thus a great potential for further industrial applications.