Identification And Characterization Of Transcriptional Factors Regulating Cellulase Gene Expression As Well As Proteins Associated With Cellulase Secretion In Trichoderma Reesei

Biomass resources represented by cellulose are a rich and renewable green resource that can be used as an effective supplement to fossil resources.Trichoderma reesei is a kind of model filamentous fungus used in industry to degrade lignocellulosic substrates.It can secrete a variety of cellulases withcellulose substrate,yielding up to 100 g/L in industrial production.As extracellular secreted proteins,the high-level extracellular secretion of cellulases usually involves processes including transcription,translation,folding modification,sorting,and vesicular transport.Previous studies have identified a number of transcription factors involved in regulating the expression of cellulase genes,including the key transcriptional activator XYR1.However,the transcriptional regulatory network of cellulase genes has not been fully elucidated,and there are still many unknown transcription factors that need to be explored.Due to its strong protein secretion ability,T.reesei has the potential to efficiently express chassis cells as heterologous proteins.However,previous studies have found that the production of many heterologous proteins in T.reesei has not reached the expected level.On the other hand,the modification of the cellulase gene transcription level did not achieve the expected yield,and it is speculated that there are speed limiting steps to further increase the cellulase yield in the subsequent folding secretion pathway.Currently,the research on the protein secretion pathway of T.reesei is at the initial stage,and the molecular chaperone proteins that assist in the efficient folding and transportation of cellulase in the protein secretion pathway are still unclear,which is a bottleneck limiting the production of cellulase and the efficient expression of heterologous proteins in T.reesei.Based on the above problems,this research thesis focuses on the screening and functional characterization of transcriptional regulators and secretory molecular chaperones involved in cellulase production in T.reesei.The main research results obtained are as follows:1.Screening and identification ofa transcriptional regulator TAM1 in Trichoderma reesei that functions as a coactivator of ARE1 in participating in regulating both ammonium assimilation and cellulase biosynthesisA transcription factor TAM1 that affects the expression of cellulase was screened and identified from transcriptome data.The homologous protein of this transcription factor in Aspergillus nidulans is known to be involved in regulating nitrogen metabolism.The growth of tam1 deficient strains in different nitrogen source culture media was determined,and the results showed that TAM1 was involved in regulating the ammonium salt utilization pathway of T.reesei.At the same time,it was found that after tam1 deficiency,the transcription level of the glutamate dehydrogenase gene gdh1 responsible for converting ammonium salt absorbed by cells into glutamine decreased.The determination of cellulase expression in tam1 deficient strains under cellulose induced conditions showed that tam1 deficiency also resulted in a decrease in the overall expression and secretion level of cellulase;Transcription analysis revealed that the transcription of the cellulase gene and the transcription activator xyr1 in the missing strain was downregulated.By measuring the phenotypes of recombinant strains overexpressing gdhl and xyr1 genes in tam1 deficient cell,it was determined that TAM1 has independent regulatory functions on cellulase gene transcription and ammonium salt absorption metabolism.Analysis of mutations in the DNA-binding domain of TAM1 showed that the regulation of TAM1 on the above genes did not depend on its direct binding to the promoter region.Furthermore,the interaction between TAM 1 and the carboxyl terminal regions of AER1 was determined through yeast two hybrid analysis.TAM1 may serve as the coactivator of ARE1 to regulate the transcription of related target genes.In summary,the nitrogen source metabolism regulatory transcription factor TAM1 identified in T.reesei can simultaneously participate in regulating the transcription of cellulase genes.2.The interactome of cellulase CBHI during its secretion process was identified,and analysis showed that thiscollection of interacting proteins is closely related to protein folding secretion pathwayAn immune coprecipitation(Co-IP)system based on protein cross-linking in vivo was established in T.reesei using the liposolubility and permeable membrane protein crosslinker DSP(Dithiobis(succinimidylpropionate).KOG function annotation and KEGG function enrichment analyses showed that this proteome is closely related to the protein synthesis and secretion pathway of T.reesei,including a variety of molecular chaperones proteins distributed in HSP70,HSP90 and 14-3-3 families.3.The HSP90 molecular chaperone HSC82 of Trichoderma reesei was involved in maintaining the normal growth and development of mycelium,and was related to the protein synthesis and secretion pathwayThe protein HSC82 annotated as the molecular chaperones of the HSP90 family was screened and identified from the CBHI interacting proteome.The hsc82 gene was knocked down through RNA interference system,and the growth phenotype of the knockdown strain was determined.It is found that HSC82 plays a very important role in maintaining the normal hyphal growth,branching and sporulation.The extracellular enzyme activity and cellulase gene transcription level of the mutant strain under cellulose induced conditions were measured,and it was found that the knockdown of hsc82 damaged the ability of T.reesei to respond to induction signals to initiate cellulase gene transcription.Fluorescence localization analysis showed that HSC82 fused to the green fluorescence protein was localized in the cytoplasm.Further,the interaction proteome of HSC82 under glucose and cellulose culture conditions was isolated and identified through the DSP cross-linked Co-IP system,and KEGG function enrichment analysis of the proteome showed that the interaction protein functions of HSC82 were enriched in exosome,protein translation,ribosome composition,genetic information processing and other functional pathways,which were closely related to protein synthesis and maturation process.Altogether,these results indicated that the HSP90 family molecular chaperones HSC82 participates in the biosynthesis and secretion of certain proteins to maintain the normal growth and development of strain,and also participates in the induction and expression of the cellulase gene of the strain.4.The 14-3-3 family molecular chaperones FTT1 and FTT2 of Trichoderma reesei were shown to play important roles in sustaining the normal growth and development of mycelium as well as the expression and secretion of cellulasesFTT1 and FTT2 annotated as 14-3-3 family molecular chaperones were screened and identified from CBH1 interaction proteome.By constructing strains that express fusion sfGFP in situ with FTT1 and FTT2,fluorescence localization analysis was performed to clarify the cytoplasmic localization of FTT1 and FTT2.The phenotypic determination results of FTT2 knockout strains showed that the absence of FTT2 alone did not affect the normal growth of the strain,only caused a slight decrease in extracellular cellulase activity and weakened stress resistance under mild environmental pressure.Since FTT1 could not obtain the knockout strain of its coding gene,the fttl and ftt2 genes were knocked down by RNA interference system.The growth phenotype test found that the interfering strain exhibited severe growth defects,and could not form branching normally during mycelial growth.Transcription level analysis revealed that the knockdown strains of fttl and ftt2 were unable to respond to induction signals to initiate the expression of cellulase genes under cellulose induction conditions,but the expression level of cellulase driven by the Ptcu1 promoter was significantly increased under glucose conditions.Similarly,knocking down fttl and ftt2 also increases the transcriptional level of cellulase genes caused by the constitutive expression of XYR1 in the Ptcu1 promoter,leading to a significant sustained accumulation of cellulase in the cell and a corresponding increase in extracellular secretion.The phenotypes of mutant strains overexpressing FTT1 and FTT2 phosphorylation substrate binding sites were determined,which showed that the phosphorylation protein binding ability of 14-3-3 family was critical to the expression of cellulase,but the related mutations did not affect the growth and development of the basic mycelium.The cross-linked interaction proteins of FTT1 and FTT2 under the conditions of glucose and cellulose were separated and identified.KEGG function enrichment analysis found that the common protein functions of FTT1 and FTT2 under the conditions of two carbon sources were concentrated in exosomes,protein translation,ribosome composition,and genetic information processing,indicating that FTT1 and FTT2 were jointly involved in the biosynthesis and secretion of some specific proteins under the conditions of two carbon sources.FTT1 also participates in some biological processes related to basic growth and development under glucose conditions.In addition,FTT1 may also be involved in response and metabolic pathways related to cellulose degradation.In contrast to the wild-type FTT1,the intracellular localization of the phosphorylated protein binding site mutant FTT1 changes,and its interacting cross-linking proteome is significantly different from the wild-type FTT1.The potential target protein obtained by subtractive analysis is closely related to the function of protein synthesis and secretion pathway.In conclusion,the molecular chaperones of T.reesei 14-3-3 family plays an important role in maintaining the normal growth and development of the strain and the expression and secretion of cellulase,and its phosphorylated substrate protein is an important target for regulating the secretion of cellulase.