Transcriptional Reprogramming Of (Hemi) Cellulase Biosynthesis And Functional Exploration Of Signal Recognition Particle In Trichoderma Reesei

Lignocellulosic biomass is the most abundant renewable carbohydrate on the planet.It is an ideal raw material for the production of second-generation biofuels and biorefined products.However,the stubborn degradation of raw materials makes the conversion of biotechnology complex and costly.The filamentous fungus Trichoderma reesei has attracted people's attention due to its strong(hemi)cellulase synthesis and secretion ability.Its mutant strain can produce up to 100 g of cellulase-rich extracellular protein per liter of culture.The powerful protein synthesis and secretion ability of T.reesei also makes it a good host for the production of recombinant proteins in industrial applications.However,the(hemi)cellulase cocktails secreted by T.reesei has a high proportion of exonuclease and endonuclease,while ?-glucosidase and hemicellulase account for a relatively low proportion,which limits the further improvement of the degradation efficiency of the(hemi)cellulase cocktails to the lignocellulosic substrate.The current research on the mechanism of high-efficiency protein synthesis mainly focuses on gene transcription in T.while there are few studies on synthetic secretion pathways.This limits genetic modification based on synthetic secretion mechanisms to further increase the expression level of endogenous and heterologous proteins.Endoplasmic reticulum translocation is the primary link in the efficient synthesis of secreted proteins.Inefficient endoplasmic reticulum translocation will greatly reduce the yield of protein synthesis.Although the classical theory believes that endoplasmic reticulum translocation is mediated by signal recognition particle through the N-terminal signal peptide of the recognition protein,studies have found that there are SRP-independent pathways for endoplasmic reticulum translocation in other species.As a model strain in filamentous fungi,the role of SRP in the efficient synthesis and secretion of T.reesei protein is worthy of our discussion.This will not only help reveal the unknown efficient translocation pathway of the endoplasmic reticulum,but also guide the development of T.reesei endogenous and heterologous protein expression systems,and help promote the application of filamentous fungal protein expression cell factories.This thesis mainly includes two aspects of research.On the one hand,it is based on the(hemi)cellulase transcription regulation mechanism.We use strain modification strategies to optimize and reconstruct the extracellular(hemi)cellulase degrading cocktails of T.reesei.On the other hand,we explored the function of important subunits of SRP complex in T.reesei.The main research work is summarized as follows:1.A strain of ?5677OExyr1 with deletion of the main cellulase gene and overexpression of the transcriptional activation regulator XYR1 was constructed.Compared with the wild-type strain QM9414,the composition of the extracellular(hemi)cellulase cocktails of this strain has significantly changed under the conditions of glucose and lactose carbon source.It has a higher level of glucosidase and a variety of hemicellulase enzyme activities,which can effectively promote the degradation of(hemi)cellulose substrates.Using the ?5677 strain as the starting strain,which deletes both endonucleases CEL5A and CEL7B and exonucleases CEL6A and CEL7A,we used the strong promoter of tcu1 to overexpress the transcriptional activator XYR1.This strain changes its extracellular(hemi)cellulase cocktails under soluble carbon sources such as glucose and lactose,and has high levels of ?-glucosidase,xylanase,and mannanase activities.Mass spectrometry identification of extracellular proteins found that the expression levels of(hemi)cellulases such as BGL1,XYN3,and MAN5A,as well as degradation auxiliary proteins such as CIP and Swollenin,were up-regulated.Under lactose culture conditions,the expression level of ?-L-arabinofuranosidase in the extracellular(hemi)cellulase cocktails of this strain was also significantly increased,and its enzyme activity was 10 fold higher than that of the QM9414 strain,reaching 8 U/mL.The extracellular fermentation broth of the strain ?5677OExyr1 under the conditions of glucose and lactose can significantly promote the release of hemicelluloses in the corn fiber.2.In the ?5677OExyr1 strain constructed in Chapter 1,we used the cbh1 strong promoter to drive the expression of the endoglucanase HiNce5 derived from Humicolo insolens under different carbon sources,and it is found that this strain has the potential as a good host for heterologous protein expression.The HiNce5 gene site-directed expression vector was constructed and integrated into the cbh1 sites of the ?5677 and ?5677OExyr1 strains respectively to realize the expression of HiNce5 driven by the cbh1 promoter.The results showed that HiNce5 was successfully expressed and secreted in ?5677 and ?5677OExyr1 strains.The expression and secretion level of HiNce5 in ?5677OExyr1 strain was significantly higher than that in ?5677,and both glucose and lactose could be used as culture carbon sources,indicating that ?5677OExyr1 strain can be used as a good chassis cell for heterologous protein expression.3.The promoter replacement strains for the genes encoding the signal recognition particle subunits SRP54 and SRP72 were constructed.Our analysis found that knockdown of srp72 did not significantly affect the growth of the strain,while knockdown of srp54 severely affected the physiological growth and cellulase expression of T.reesei.But it did not completely destroy the synthesis and secretion of cellulase.The srp54 and srp72 promoter replacement strains of the signal recognition particle subunit encoding gene srp54 and srp72 were constructed by the promoter replacement system in response to copper ions.The study found that compared with wild-type QM9414,srp72 knockdown did not significantly affect the growth of the strain,while srp54 knockdown resulted in a significant impact on the growth and biomass accumulation of T.reesei,and the integrity of the cell wall was severely damaged.In addition,the main cellulase expression and extracellular cellulase activity were significantly reduced after srp54 knockdown,but the cellulase secretion was not completely destroyed.The mature form of the main exonuclease CBH1 was detected both inside and outside the cell,suggesting that there may be a SRP-independent pathway to assist the translocation of cellulase-encoded mRNA to the endoplasmic reticulum.