Study On The Role Of The Post-translational Endoplasmic Reticulum Translocon Subunits In Efficient Synthesis And Secretion Of Cellobiohydrolase Ⅰ By Trichoderma Reesei

The filamentous fungus Trichoderma reesei has a strong capacity for cellulase synthesis and secretion,and the cellulase produced is widely used in various fields of industrial production.T.reesei is also used as a chassis cell for the secretion of heterologous proteins.Therefore,the analysis of the efficient cellulase synthesis and secretion mechanism of T.reesei can not only provide a theoretical basis for the further improvement of endogenous cellulase production through genetic modification,but also provide effective reference for the efficient production of heterologous proteins.The cellulase exonuclease CBHI is the main component of the extracellular enzyme system of T.reesei,reaching 60%of the total extracellular protein production.The current studies on the efficient synthesis and secretion mechanism of CBHI are mainly focused on the efficient transcriptional regulation of genes,and less at the level of protein secretion pathway.Translocation across the ER membrane is the "entry" and rate-limiting part of the secretory pathway.Efficient and accurate endoplasmic reticulum translocation is a prerequisite for normal cellular physiological functions.The mechanism of efficient translocation of T.reesei cellulase and even filamentous fungal proteins is still unclear,which greatly limits the study of genetic modification of this link to improve the production of endogenous cellulase and heterologous proteins.The translocation of nascent polypeptides to the endoplasmic reticulum is mainly divided into two ways:cotranslational and post-translational.Based on the results of our Lab’s previous studies,we hypothesized that cellulase CBHI takes a post-translational approach to translocate to the endoplasmic reticulum.In this thesis,we investigated the role and mechanism of posttranslational endoplasmic reticulum translocation channel subunits in the efficient synthesis and secretion of CBHI.The main contents of this thesis and the main results obtained are as follows:1.The Sec72 subunit plays a specific role in the efficient synthesis and secretion of cellulase CBHI in Trichoderma reesei.Two genes encoding post-translational endoplasmic reticulum channel subunits,sec71 and sec72,were knocked out in Trichoderma reesei,and the analysis revealed that the deletion of Sec71 severely affected the growth and sporulation ability of the strain,while the deletion of Sec72 barely affected the growth and sporulation ability of the strain.However,both deletions resulted in severely impaired expression and secretion of the major cellulase CBHI,suggesting that the post-translational translocation channel plays an important role in the synthesis and secretion of CBHⅠ.Combined enzyme activity assay,SDS-PAGE and Western blot analysis revealed that the deletion of Sec72 led to the complete elimination of extra-cellular secretion of CBHⅠ,but the secretion of three other major cellulases,cellulose exon-uclease CBHⅡ and endonucleases EGⅠ and EGⅡ,was not completely eliminated.It indicates that Sec72 plays an important role in the efficient translocation and secretion of CBHⅠ specifically.This result also implies that the translocation of CBHⅠ to the endoplasmic reticulum is mediated through the post-translational translocation channel.2.The specific action of Sec72 on cellulase CBHⅠ occurs at the signal peptide level.To investigate whether the recognition effect of Sec72 on CBHⅠ occurred at the signal peptide level,the signal peptide of CBHⅠ was replaced with the signal peptide of cellulase CBHⅡ,EGⅠ and EGⅡ in the sec72 knockout strain,respectively,which were not dependent on Sec72.Analysis of CBHⅠ enzyme activity assay and Western Blot assay of extracellular fermentation broth of the replacement strain showed that the replacement of signal peptide greatly restored the synthesis and secretion level of CBHⅠ from zero to 50%-70%of the level of wild-type strain,indicating that the signal peptide of CBHⅠ is the main recognition region of Sec72.At the same time,the same signal peptide replacem ent was performed for CBHⅠ in the wild-type strain,and the analysis revealed that the synthesis and secretion level of CBHⅠin the wild-type strain decreased to 50%-70%of the original level,indicating that CBHⅠ own signal peptide has high efficiency,which is important for the efficient synthesis and secretion of CBHⅠ.The comparative analysis of the synthesis and secretion levels of CBHⅠ after signal peptide replacement in Sec72-deficient strain and wild-type strain showed that the levels were very close to each other,which indicated that the specific effect of Sec72 on CBHⅠ occurred entirely at the signal peptide level.3.The cytoplasmic molecular chaperone Ssb1 plays an important role in Sec72-mediated efficient translocation and secretion of CBHⅠ.In vitro biochemical studies reported that the cytoplasmic molecular chaperones Ssa1 and Ssb1 of Saccharomyces cerevisiae interact with Sec72 to promote the anchoring and translocation of nascent proteins to the endoplasmic reticulum.To further elucidate the role of Sec72 in the efficient translocation and secretion of CBHⅠ in Trichoderma reesei,we successfully obtained knockdown/knockdown strains of Ssbl and Ssa1,respectively.ssb1 knockdown did not cause serious damage to strain growth,but resulted in the complete elimination of CBHⅠ synthesis and secretion,indicating that the molecular chaperone Ssb1 plays an important role in the efficient synthesis and secretion of CBHⅠ.When the signal peptide of CBHⅠ was replaced by the Sec72-independent EGⅡ signal peptide in the ssb1 knockdown strain,the level of CBHⅠ synthesis and secretion was partially restored,indicating that the signal peptide is a major region of Ssbl recognition of CBHⅠ.