Deciphering The Cellulase Production Mechanism Of Trichoderma Reesei CU7-4 And Construction Of The Engineering Strain With Optimized Lignocellulases

Trichoderma reesei is an industrial filamentous fungus currently used for cellulase production and also a model strain for expression and secretion studies of cellulase.The T.reesei strain was first isolated from decaying military canvas in World War II,and the original strain was named QM6a.After that,a series of mutant strains with significantly increased cellulase production were obtained by multiple rounds of mutagenesis,among which RUT-C30 is a widely used high-yield cellulase industrial strain,and the cellulase activity of RUT-C30 is 15-20 times higher than that of QM6a.Although most of the studies of T.focus on the cellulase-producing mechanism,there are still a large number of blind spots,which due to the complexity of lignocellulose-degrading enzyme system,the relationships between enzymesynthesis/secretion and global tunning of cells,especially many genes with unknown functions exist in the genome.In addition,although the lignocellulose-degrading enzymes secreted by T.reesei contains a large amount of cellulase,the enzyme system is still unbalanced for degrading different lignocelluloses.In particular,the high cost of cellulase still hinders the conversion of biomass resources.Therefore,further improvement of the cellulase production capacity of strains and in-depth study of cellulases composition and functions of novel genes would be helpful to elucidate the secretion mechanisms of cellulase system and construct engineering strains with optimized lignocellulose-degrading enzymes,which would thereby improve the conversion efficiency of lignocellulosic biomass,reduce the cost of cellulase in industrial scale and facilitate the economic feasibility of biomass conversion.In the previous study in our laboratory,a cellulase hyperproduction mutant strain CU7-4,whose cellulase production was 3～4 times higher than that of T.reesei RUT-C30,was obtained by deep mutagenesis on the basis of RUT-C30.In this study,CU7-4 was taken as the research object to analyze extracellular cellulases composition by secretome identification,characterize the key functional genes through genome and transcriptome analysis,analyze the cellulase production under the carbon catabolite repression(CCR)condition,and,especially,construct the engineering strain equipped with optimized lignocellulose-degrading enzymes.The main research contents and results are as follows:1.Analysis of the high efficiency mechanism of lignocellulosic degradation by the cellulase system of the mutant CU7-4Firstly,the extracellular enzymes of CU7-4 and RUT-C30 were applicated to saccharify the lignocellulose substrates acid-pretreated(ACR)and delignified corncob residues(DCR),and it was found that CU7-4 increased the saccharification efficiencies by 20%-21%toward both substrates compared to RUT-C30,which indicated that the extracellular enzyme system of CU7-4 has more efficient ability to degrade lignocellulose than RUT-C30.Then,the comparative proteomics was employed to analyze the differences between the secretomes of CU7-4 and RUT-C30,and the results showed that the proportion of cellulase components changed significantly,such as the remarkable increase of endoglucanase.Five potential target proteins were screened by analyzing the ten of proteins having a large difference between CU74 and RUT-C30 in secretion and in response to the transcriptional activator Xyr1:three small secreted cysteine-rich proteins SSP1,EPL1,and CUT1,and two β-glucosidases Cel3H and Cel3F.The gene knockout study exhibited that the deletions of two small secreted proteins SSP1 and EPL1 led to an increase in the ability to degrade different lignocellulose substrates,especially the degradation of low lignin content substrates ACR,DCR and NPCS(alkali pretreatment of corn stover).Among them,the saccharification efficiency of the SSP1 deletion strain towards NPCS was increased by 37.1%compared with that of the parental strain.This suggests that SSP1 and EPL1 play an important role in the hydrolysis of lignocellulose by T.reesei.The studies of gene knockouts and overexpressions of Cel3H/Cel3F showed that overexpression of Cel3F could lead to improvement of cellobiohydrolase and β-glucosidase activities,indicating that Cel3F might play an important role in cellulase production of T.reesei.Furthermore,single Cel3F protein was added to the fermentation broth of T.reesei to study its effect on saccharifying ACR and DCR,and the results showed that the increment of the saccharification efficiency caused by the addition of Cel3F of 10%-protein content was equivalent to that of RUT-C30 fermentation broth of more than 50%-protein content,which suggested that Cel3F could significantly improve the lignocellulose degradation efficiency by T.reesei cellulase system.2.The molecular mechanism of the hyperproduction of cellulases by CU7-4Firstly,the transcriptomics strategy was employed to compare the differences between the mutant strain CU7-4 and RUT-C30 at the transcriptional level,and it was found that most of the differential expressed genes(DEGs)were enriched in the extracellular protein genes,especially carbohydrate hydrolases(Cazymes)related to the degradation of lignocellulose,among which β-glucosidase genes were highly enriched,indicating that they may also play an important role in the high cellulase production.Further analysis of the DEGs distribution in the KEGG pathways showed that the transcriptional levels of the three subunits TTDA,TFIIH2 and MANT1 of the transcription factor TFIIH were significantly upregulated.The expression in different degrees of genes ttda,tf2h2 and mantl of these three subunits were carried out,and it was found that their low expression had an impact on the transcription of cellulase genes in T.reesei,especially,the low expression of TTDA significantly reduced the gene transcription and secretion levels of cellulases,while the enhanced expression of the three genes did not have a significant effect on the expression of cellulases in T.reesei.Since TFIIH is both a basal transcription factor and a conserved protein involved in nucleotide excision repair(NER),it was speculated that UV mutagenesis may have stimulated the NER pathway of CU7-4,resulting in the increased expression of the three subunits and thus provide the possibility of cellulase hyperproduction.However,this change did not have a direct effect on cellulase expression.Then,the mutations of CU7-4 were analyzed by genome resequencing.A total of 59 single nucleotide polymorphism(SNP)mutations and 21 small segment insertion/deletion(InDel)(all located in the intergenic region)were detected.Furthermore,the deletion of the single gene containing the 12 SNPs causing nonsynonymous mutations was carried out,respectively,and it was found that the knockout of one of the genes,tre130442(encoding protein INO80 subunit,a chromatin remodeling complex),seriously hindered the growth of T.reesei and significantly reduced cellulase production,and the transcriptional level analysis of the cellulase genes showed that the effect of this gene on cellulase was not caused by the mycelial growth defect.Besides,the single nucleotide mutant strain of this gene led to negative effect on growth to some degree.Combined with CU7-4 transcriptome data analysis,it is speculated that the effect of this gene on CU7-4 growth might be the result of a combination of SNP,reduced expression and participation in DNA repair.In addition,genome sequencing also found three large structural variants in CU7-4,including two inverted fragments and one duplicate fragment,which involved 400 genes.Among them,the genes with significantly different transcriptional levels contains glycoside hydrolases,transcription factors,transporters and so on,these genes may also play a potentially important role in the hyperproduction of cellulase in CU7-4.3.Study on the derepression of carbon catabolite repression of the mutant CU7-4By culturing the mutant strain CU7-4 in different carbon sources and detecting its cellulase production capacity,it was found that its cellulase activity in glucose was comparable to that of RUT-C30 in Avicel,and the cellulase activity of CU7-4 was 8.3-fold higher than that of RUTC30 under cellobiose condition.Moreover,CU7-4 could still produce cellulase that was equivalent to that of the fermented under the sole Avicel when the glucose analogue 2deoxyglucose is added to Avicel,while RUT-C30 had barely cellulase production,which indicated that the CCR pathway of CU7-4 is damaged,and thus CU7-4 has a higher degree of CCR derepression ability compared to RUT-C30 that partially alleviated the CCR effect.At the same time,CU7-4 has a decrease in glucose consumption compared to RUT-C30.Further analysis of the transcriptional level of CCR pathway-related genes in CU7-4 under glucose condition showed that the gene expression levels of glucose transporter Gltl,the transcription factor BglR regulating Gltl,and the transcriptional repressor Crel that dominates CCR were significantly downregulated.In addition,by adding different concentrations of glucose to Avicel in the fermentation,we found that the resistance of CU7-4 to glucose inhibition was enhanced compared to RUT-C30,and cellulase activity reached the highest level of 8.75 IU/mL when 10 mM glucose was added,which was 19%higher than that under the sole carbon source of Avicel.These results suggested that CU7-4 has a higher degree of deCCR effect and can produce high yield of cellulase in the presence of glucose.4.Construction of the engineering strain with optimized lignocellulose-degrading enzymes by overexpressing endoglucanase and β-glucosidaseIt was found that endoglucanase and β-glucosidase were significantly improved in CU7-4 and also were key components of the efficient lignocellulose-degrading enzyme system,thus these two cellulases were selected as target proteins for cellulase system optimization.Firstly,by fusing the activation region of the inducible promoter Pcbh1to the core region of constitutive promoter Pcdna1,a novel hybrid promoter Pcc was constructed,which could efficiently promote gene under both glucose and Avicel conditions.Especially,the activity of Pcc was 1.6 and 1.8 times of Pcbhl and Pcdnal under Avicel condition,respectively.After that,Pcc was used to expressing the endoglucanase gene eg2 and β-glucosidase gene bglA,which significantly increased EG and BG activities,respectively,and thereby resulting in 150%and 85%enhancement in total cellulase activities,respectively.Furthermore,the two genes eg2 and bglA were co-overexpressed conducted by Pcc,and the total cellulase activity of the resulted strain was increased by 178%compared with the original strain.Besides,the saccharification efficiencies of the engineering strain toward ACR and DCR was increased by 130%～140%.Thus,an engineering strain possessing optimized lignocellulose-degrading enzyme system was constructed,which not only provides strain resources to reduce the cost of enzyme production,but also contributes to the economically feasible bioconversion of lignocellulosic resources.