Application Of CRISPR/Cas9 Technology For Improving Cellulolytic Enzymes And Expressing Heterologous Protein In Trichoderma Reesei

The filamentous fungus Trichoderma reesei is the major industrial fungus for cellulase production and is also a good host for heterologous protein expression.However,factors such as relatively low gene knockout efficiency and relatively few selection of genetic markers severely limit the progress of species improvement and synthetic biology of T.reesei.CRISPR/Cas9 technology is an efficient gene editing technology,although developed in T.reesei,the application of it for improving T.reesei still needs to be continuously expanded.Celulase expression secretion of T.reesei is tightly regulated,where Xyr1 is the major transcriptional activator,and overexpression of this factor contributes to elevate cellulase production.Therefore,exploring the application of CRISPR/Cas9 technology in T.reesei by targeting Xyr1 will facilitate its cellulase and heterologous protein production.At first,this study used Cas9 vector integratied into the T.reesei genome,selecting Ade2(a carboxylase in the purine synthesis and loss of this enzyme produces red colonies)encoding gene as the locus to express a highly active Xyrl mutant Xyr1-A824V to explore the effect of CRISPR/Cas9 technology on improving T.reesei.Furthermore,to avoid Cas9 genome retention simultaneously with the recycling of screening markers,this study used autonomous replicating plasmids to express Cas9 and sgRNA free from outside the genome.The transcriptional repressors Cre1 and Acel encoding gene loci were selected for knockdown verification and expression of Xyr1-A824V,respectively,to improve the cellulase production of T.reesei.Lastly,glucose oxidase of A.niger was heterologous expressed using the established cas9-non-genomic integrated CRISPR/Cas9 technology.The research contents are described as follows:(1)Expression of Xyr1-A824V by Cas9-integrated into the genome in T.reesei.Firstly,the Cas9 expression vector pgCas9DR and the Xyr1-A824V expression cassette(with Pcdna1 as the promoter for expression)targeting the Ade2 gene locus(the donor DNA)were constructed,respectively.The sgRNA driven by the 5S rRNA promoter targeting ade2 was also synthesized.Then,these three elements were simultaneously transformed into T.reesei.After genomic PCR validation and plate chromoanalysis,the transformants failed to amplify Ade2 genes on the genome,could not grow on not added adenine plates and the colonies were dark red on adenine-uncontaining plates,showing that the strain construction was successful,named as QA2Xc.Cellulase analysis under cellulose-induced condition found that total cellulase(FPA)and xylanase activities of QA2Xc were increased by 5%(1.77 U/mL)and 1.5-fold(456.14 U/mL),respectively.In addition,since the PcdnaI promoter is a constitutive promoter,the enzyme production of the strain under glucose conditions was further tested.The FPA and xylanase activity of QA2Xc increased 9-fold(0.93 U/mL),15-fold(2.10 U/mL),indicating that cellulase and xylanase genes were activated by Xyr1-A824V driven by Pcdna1 on under glucose condition.Therefore,the initial improvement of the T.reesei cellulase production was achieved by using CRISPR/Cas9 to delete Ade2 gene and express Xyr1-A824V.(2)Cas9-nongenomic integrated CRISPR/Cas9 system for expressing Xyr1-A824V.In order to avoid toxic effects of Cas9 integrated into the genome and facilitate the reuse of genetic markers,this study used an AMA1 autonomous replication plasmid free from the genome to express Cas9.And the genetic marker was constructed into the plasmid together with sgRNA.Firstly,the pFC33ptrA was constructed from the AMA1 autonomous replication plasmid pFC330 containing Cas9 linked the ptrA resistance gene.Meanwhile,for simultaneous multiple sgRNA expression,a 5S rRNA promoter-driven multiple sgRNA expression cassette sg5stRNA containing the tRNA interval was also constructed in this study.Then,the carbon metabolic repressor Crel encoding gene was targeted for knockout.Two targeted protospacer of the Cre1 encoding gene were designed and ligated into sg5stRNA by fusion PCR,followed by enzyme digestion into pFC33ptrA to construct the CRISPR/Cas9 vector pFC33ptrA-5stsgRNA(cre1*2).This vector was transformed into T.reesei,then genomic PCR and cellulose plate analysis demonstrated that the Crel gene was successfully knocked out.Since absence of Crel caused a large effect on strain growth,the cellulase-specific repressor Acel encoding-gene was further replaced in the genome locus to express Xyrl-A824V.Similar to the Crel knockout method,pFC33ptrA-5stsgRNA(ace1*2)for the Accl gene,and the Xyr1-A824V expression cassctte(donor DNA)was constructed using Pcdnal and PgpdA,respectively,and then co-transformed the T.reesei,and the transformants were named QGA1Xg and QGA1Xc,respectively.Further,under cellulose-induced conditions,cellulase total activity(FPA,2.82 U/mL)and xylanase activity(721.39 U/mL)of QGA1Xg were increased by 1.3-fold and 11-fold.The FPA(2.60 U/mL)and xylanase activity(363.77 U/mL)of QGA1Xc were increased by 1.1-fold and 5-fold.Then the enzyme production of the strain under glucose conditions was further tested.The FPA(0.13 U/mL)and xylanase activity(184.40 U/mL)of QGA1Xg were increased by 0.6-fold and 21-fold.The FPA(1.61 U/mL)and xylanase activity(343.81 U/mL)of QGA1Xc were increased by 17-fold and 41-fold.Meanwhile,after subgrowing of the engineered strain,the CRISPR/Cas9 vector was lost from the cells and the resistance traits were also restored.Cas9 non-genomic integrated CRISPR/Cas9 technology was successfully constructed in T.reesei and cellulase production was improved by knocking down the Ace1 locus and overexpressing Xyr1-A824V.(3)Heterologous glucose oxidase was expressed in T.reesei using the Cas9 nongenomic CRISPR/Cas9 technology..Firstly,the sgRNA expression cassette 5stsgRNA(cbh1*2-cbh2*2)of two major cellulase component genes cbh1 and cbh2 was designed and then digested into pFC33ptrA to construct the CRISPR/Cas9 vector pFC33ptrA-5stsgRNA(cbh1*2-cbh2*2)to knockout cbhl and cbh1 genes.The A.niger glucose oxidase(GOX)expression cassette(donor DNA integrated into cbh1)using the cbh1 promoter and terminator,was co-transferred to T.reesei with the CRISPR/Cas9 vector,and the transformant was named QGOD.Genomic PCR and transcriptional analysis showed that both cbhl and cbh2 genes of the QGOD strain were knocked out,and the glucose oxidase gene gox was integrated at the cbh1 locus and transcribed at high levels.The colonies of the QGOD strain on the o-dianisidine plate presented a red-brown halo,indicating the expression and secretion of GOX.The maximum activity of glucose oxidase in the fermentation broth reached 58.31 U/mL,by using lactose as the induced carbon source.Therefore,glucose oxidase encoding gene of A.niger at the cbh1 genomic locus was successfully heterologously expressed in T.reesei with deletion of two genes cbh1 and cbh2 by CRISPR.