| Lignocellulose is the most abundant biomass resource on earth.Cellulases can efficiently degrade lignocellulose into fermentable sugar.The sugar can be converted into various chemicals or fuels,which is of great significance to alleviate the problems of resources and environment faced by human society.Filamentous fungi represented by Trichoderma reesei,Penicillium oxalicum,Aspergillus niger and Myceliophthora thermophila can efficiently synthesize and secrete extracellular cellulases,and are the main sources of cellulases in industrial production.The synthesis of cellulase is mainly controlled at transcriptional level.Many sequence-specific transcription factors（TFs）participate in the activation or repression of cellulase genes transcription by directly binding to the promoter regions of target genes.The chromatin structure of the cell also affects the expression of target genes.Exploring the key regulatory proteins of cellulase gene expression,especially the function of transcriptional activators,and in-depth study of the complex interaction patterns between transcriptional activators and chromatin-modifying enzymes will allow us to gain a deeper understanding of cellulase gene expression.It will provide a reasonable target for the molecular transformation of cellulase production strains and improve the production efficiency of strains,which has profound theoretical significance and important practical significance.In this paper,taking the important cellulase-producing strain P.oxalicum as the research object,starting from the functional study of the Hap5 subunit of the transcription activator HAP complex,we explored the molecular mechanisms of cellulase gene expression regulated by the HAP complex and the chromatin remodeling complex RSC.The results of the research are as follows:1.Study on the mechanism of HAP complex regulating cellulase gene expression in P.oxalicumP.oxalicum HAP complex composes Hap2,Hap3 and Hap5 subunits.The P.oxalicum Hap5 subunit（PoHap5）was characterized by its biological roles including mycelial growth,transcription of cellulase genes,and extracellular glycoside hydrolase synthesis.The PoHap5 is positioned in the nucleus.Its lacking caused the mutant strain（Δhap5）spread their colonies more slowly,and the conidium synthesis was impaired.In the Ahap5 mutant,the expression of three important transcription factors BrlA,FlbC,and StuA encoding genes in the center regulatory pathway of spores was downregulated remarkably.It is suggested that HAP complex indirectly regulates synthesis of conidia by affecting the regulation pathway of sporogenesis center.Compared to the starting strain,Δhap5 mutant decreased cellulase and extracellular protein secretion,the transcription level of main cellobiohydrolase gene cbh1 and endoglucanase gene eg1 was significantly down-regulated,suggesting HAP complex affected the synthesis of cellulase by regulating the expression of cellulase encoding genes.In the experiments of tandem affinity purification（TAP）and tandem mass spectrometry（LC-MS/MS）（TAP-MS）using PoHap5 as the bait,10 subunits（Sthl,Rsc8 Arp9,Sfhl,Snf5,Ssr3,Arp4,Rsc1,Ssr4,and Rsc7）of a known chromatin remodeling complex RSC were observed,showing that chromatin remodeling complex RSC is as a cofactor of HAP complex.A possible pathway for the regulation of cellulase genes expression by the HAP complex is proposed:The HAP complex binds to the CCAAT sequence on the cellulase gene promoter region and recruits the chromatin remodeling complex RSC as a cofactor,which utilizes the energy of ATP to change the chromatin structure,exposing the RNA Polymerase II binding site on DNA,and then activating cellulase gene transcription.2.Functional studies of RSC chromatin remodeling complex subunits Sthl and FaiR.It was determined that the Sthl subunit（PoSthl）of the chromatin remodeling complex RSC in P.oxalicum is the essential gene of cell survival.Its absence will lead cell death.A protein（PDE04776）of an unknown function was found during TAP-MS with PoSthl as a bait protein.PDE04776 is a filamentous fungi-specific protein,especially in Aspergillus and Penicillium spp..Among the PDE04776 interacting proteins,the most abundant proteins are the homologous proteins that have been reported to chromatin remodeling complex SWI/SNF or RSC subunits,including Rsc8,Sfh1,Npl6,Snfl2p,Arp7,Rsc9p,Ssr4,Rscl,Sth1,Snf21.Therefore,the PDE04776 is named FaiR（filamentous fungi-specific accessory interactor of RSC）.The expression of gene encoding P.oxalicum FaiR（PoFaiR）is a constitutive expression.The deletion mutant of PoFaiR（ΔfaiR）showed growth defect on the agar containing glucose,mannose and other different carbon sources,but grew well on the cellulose agar.Cellulase gene expression was upregulated in the ΔfaiR mutant,and the secretion of extracellular cellulase and total protein increased.The ΔfaiR mutant was not sensitive to carbon catabolite repression（CCR）.In the shake flask fermentation with 2%microcrystalline cellulose as the carbon source,adding glucose could promote the expression of cellulase gene and cellulase synthesis in ΔfaiR mutant.When 0.5%,1%,and 2%glucose were added,the expression levels of exonuclease gene cbhl in ΔfaiR were 13.1,21.4,and 32.8 times higher than those in the starting strain,respectively.In the presence of glucose,the absence of FaiR significantly up-regulated the transcription of genes encoding transcriptional activators ClrB and XlnR,and down-regulated the transcription of genes encoding transcriptional repressor CreA,indicating that FaiR indirectly participates in CCR by controlling the expression of important transcription factors. |
