Identification of Transcription Factors Controlling Expression of Cellulolytic Enzymes in Trichoderma Reesei and Development of Robust Strains for Improved Cellulase Production

Author:Meng Qing Shan

Supervisor:zhao xin qing bai feng wu


Degree Year:2019





As a renewable resource,lignocellulosic biomass is abundantly available on the earth,which is composed mainly of cellulose,hemicclluloses and lignin.Degradation of the cellulose component by cellulases into glucose as feedstock for microbial culture and fermentation to produce biofuels and bio-based chemicals not only alleviates heavy dependence on fossil resources such as crude oil,but also benefits the environment for sustainable socio-economic development.However,the high cost of cellulases produced through microbial fermentation makes the glucose production too expensive for such a purpose,which ultimately limits the utilization of lignocellulosic biomass for the biorefinery.Filamentous fungi are capable of degrading lignocellulosic biomass,in which Trichoderma reesei has been intensively studied for cellulases production with a focus on understanding of molecular mechanism underlying its cellulases production for strain development through genetic engineering,and on the other hand the optimization of the ratio of its cellulolytic enzymes for their more efficient synergy in degrading the cellulose component.T.reesei Rut-C3()was previously used for cellulases production in industry,and has been used as the model strain for fundamental research since it was isolated.Now,it is frequently selected as the host strain for genetic modifications to develop robust strains for cellulases production.In this study,T.reesei Rut-C30 was engineered with artificial zinc finger proteins(AZFPs)library,and mutants T.reesei Ml and M2 with improved cellulases production were screened.Compared to the parent strain,the filter paper activity(FPase)of T.reesei M1 and M2 increased 100.8%and 53.2%,respectively,total extracellular proteins produced by T.reesei M1 increased 69.1%,and endo-β-glucanase(C.MCase)activity produced by T.reesei M2 increased 64.2%.Furthermore,the DNA sequences of AZFP-transcription factors(AZFP-TFs)in T.reesei M1 and M2 mutant were identified,respectively,which were inserted into the same loci between TrireRUTC30:4597 and TrireRUTC30:67627 in the T.reesei genome,suggesting that improved cellulases activities in the mutants were indeed due to the expression of AZFP-TFs.RT-qPCR analysis showed that expression of major genes encoding cellulolytic cellulases was substantially up-regulated in both the mutants,but different expression profiles were observed for genes encoding transcription factors:the transcription repressor ace1 was down-regulated in both mutants,but the transcription activator xyr1 was up-regulated only in T.reesei M1.These results demonstrated that AZFP-TFs exert diverse regulation on cellulases production in T.reesei.Transcription analysis for target genes of the AZFP-TF in T.reesei M2 revealed down-regulation of the putative gene TrireRUTC30:10530(Trctfl),and a deletion mutant was thus constructed for further studies.An increase of 43.8%in cellulases production was detected in the deletion mutant,and on the other hand when the gene was constitutively overexpressed in T.reesei Rut-C30 under the control of the strong promoter pdc1,cellulases production was significantly repressed.Comparative transcriptome analysis indicated that the deletion of Trctfl up-regulated the trantscription of the positive regulator VIB1 and down-regulated the negative regulator RCE1,which consequently up-regulated the transcriptors XYR1 and ACE3 for activating the expression of genes encoding cellulolytic enzymes.As a result,Trctf1 was identified as a repressor in T.reesei Rut-C30 for cellulases biosynthesis,which is significant for elucidating regulation mechanism underlying cellulases biosynthesis in T.reesei for strain development through rational design.Meanwhile,AZFP engineering has been validated to be an effective strategy for identifying functions of putative genes in the genome of T.reesei.The AZFP-TF inserted into T.reesei M2 consists of the sequence-specific DNA-binding domain and yeast-based Gal4 activation domain.In T.reesei,its cellulases synthesis is mainly under the control of the Ga14-type transcrition regulator Xyr1.Therefore,a novel artificial chimeric transcription factor AZFP-M2-Xyrl AD was designed to investigate its effect on cellulases production in T.reesei.Azfp-M2-ga14 from T.reesei M2 and Azfp-M2-xyr 1 AD were inserted into the xyn3 gene locus in T.reesei TU-6 independently to develop mutants T.reesei QS1 and QS2.Compared to the parent strain,the FPase of T.reesei QS1 and QS2 increased 39.4%and 73.7%,respectively.Analysis of the transcription profile provided evidence that expression of genes encoding major cellulolytic enzymes and accessory proteins were significantly up-regulated in both mutants,but the expression of transcription factors varied in T.reesei QS1 and QS2,which suggested that the two AZFP-TFs play different roles in regulating cellulases synthesis in T.reesei.Moreover,saccharification of alkali pretreated corn stover and Jerusalem artichoke stalk with crude enzyme produced by T.reesei QS2 showed increases of 97.9%and 14.0%as well as 90.2%and 8.2%in glucose yield compared to that achieved with crude enzyme produced by the parent strain and T.reesei QS1.These results demonstrated that AZFP-M2-Xyrl AD is more efficient than AZFP-M2-Ga14 for regulating cellulases production in T.reesei.The ratio of cellulases produced by T.reesei is not optimal for efficient hydrolysis of the cellulose component in lignocellulosic biomass,particularly an insufficient production of endoglucanase.Although cellulases formulation has been practiced,in vivo production of cellulases cocktail with an optimized ratio for cellulolytic enzymes is preferred.T.reesei QS305 with high endoglucanase activity was thus developed from T.reesei Rut-C30 by replacing the transcription repressor gene ace1 with the coding region of endoglucanase gene egll.Compared to the host strain,T.reesei QS305 showed 90.0%and 132.7%increase in the activities of FPase and endoglucanases under flask culture conditions.When cellulases production by T.reesei QS305 was performed in the 5-L fermentor,cellulases activity of 10.7 FPU/mL was achieved at 108 h,75.4%higher than that produced by T.reesei Rut-C30.Moreover,cellulases produced by T.reesei QS305 were more efficient for hydrolyzing pretreated corn stover and Jerusalem artichoke stalk.This work provides a basis for understanding the regulatory network of cellulases biosynthesis by T.reesie,and thus enabling more fine-tuned manipulations with the species for strain development through genetic engineering to improve its cellulases production.