Construction Of Trichoderma Reesei Expression Host Strains With Low Cellulase Background

The filamentous fungus Trichoderma reesei is an important cellulase industrial production strain.Due to its excellent enzyme-producing properties,more and more studies have been carried out in recent years to use it as an expression host for heterologous genes.T.reesei produces a large number of endogenous cellulases,including cellobiohydrolases(exoglucanases)and endoglucanases.When using T.reesei to express foreign genes,they will form a high cellulase background,and their expression may lead to occupation of related resources such as cell transcription,translation,and secretion.In this experiment,CRISPR/Cas9 technology was combined with RNAi interference technology to knock out and interfere with the main cellulase genes in T.reesei to construct a low-cellulase expression strain of T.reesei.The experiment also found that using a low cellulase background strain as an expression host can significantly increase the expression level of some heterologous genes.The following work has been performed in this study:1.Using the CRISPR/Cas9 technology to knock out the major cellobiohydrolase cbh1 gene in T.reesei;in the meantime,RNAi was used in combination to silence the expression of endoglucanase eg2 gene in T.reesei.CRISPR/Cas9 technology was used to assemble Cas9/gRNA complex in vitro and transform T.reesei to knock out the major cellobiohydrolase cbh1 gene in conjunction with RNAi technology to silence the major endoglucanase eg2 gene in order to construct a low-cellulase background expression system of T.reesei.Using this method,a strain SUS6 with a significantly reduced cellulase expression pattern was obtained.In this transformant,the expression of cbh1 completely disappeared,while the transcription level of the eg2 gene was 98% lower than that of the starting strain SUS5.2.Expression of the exogenous gene NfBgl3 A in SUS6 strain.Using SUS6 as the host to express the exogenous gene NfBgl3 A,the beta-glucosidase activities of the two representative transformants were 172.4 and 79.3 U / mL,respectively,which were much higher than the beta-glucosidase activities from two representative transformants(11.6 and 31.9 U / mL)using the starting strain(high cellulase background)as the host.However,the expression of mannanase derived from Aspergillus niger did not significantly increase.Therefore,constructing a strain with a low cellulase background as an expression host could also selectively increase the expression level of certain heterologous genes.3.Applying CRISPR/Cas9 technology to knockout the main secreted proteins xylosidase(xyl3A)and cellulase(cbh2 and eg1)genes based on SUS6 strainBased on the SUS6 strain,first the pyr4 selection marker of T.reesei was removed by 5-FOA reverse selection,and then the xyl3 A gene was knocked out by intracellular expression of Cas9 nuclease and gRNA.A transformant strain SUS7 with the knockout of xyl3 A gene was obtained.Then using SUS7 strain as the host strain,the pyr4 selection marker was knocked out.Then,using CRISPR/Cas9 technology,the in vitro assembled Cas9/gRNA complex was combined with the homologousrecombination arm and jointly transformed into T.reesei to knock out the cbh2 and eg1,respectively.This led to a further construction of the expression hosts of T.reesei with even lower cellulase background.4.Exploration of new CRISPR / Cas9-based editing methods in T.reesei.As the third-generation genome editing technology,the CRISPR/Cas9 system has high-efficiency gene editing capabilities.However,when we applied it to T.reesei,we found that its efficiency was still very low.In response to this problem,we have tried to improve the CRISPR/Cas9 system from various perspectives,such as tmodification of the promoter that drives gRNA,mutation of Cas9 protein,addition of non-homologous DNA,addition of small molecular substances,and the use of heat stress conditions,etc.,in order to improve the gene editing efficiency of CRISPR/Cas9 system in T.reesei.In summary,in this study,various methods were comprehensively used to construct expression host strains of T.reesei with low cellulase background.Using a strain in the construction process as an expression host could significantly increase the expression level of certain heterologous genes.In addition,we have carried out useful explorations on some new editing methods.