Engineering Of The Highly-Efficient Promoters In Trichoderma Reesei And Their Applications In The Expression Of Lignocellulose-degrading Enzymes

Trichoderma reesei is widely used in the production of cellulase and other heterologous proteins in industry.It has been reported that the yield of extracellular cellulases in T.reesei can exceed 100 g/L.As a safe host in food industry,in T.reesei also has the advantage of relatively low fermentation cost.In order to realize the production of industrial enzymes,food proteins and pharmaceutical proteins in T.reesei,it is necessary to construct an efficient protein production system.As the first step in protein production,the transcription stage is very important,in which strong promoters are the key to achieving high levels of gene transcription.Therefore,in this thesis,the strong promoter Pcdnal and the inducible promoter Pcbh1 in T.reesei were engineered respectively,to obtain more efficient promoter mutants for the expression of target proteins.The main results of this research are as follows:1.Engineering of the promoter PcdnalGene cdnal,whose promoter is commonly used for recombinant protein production in T.reesei,encodes a small unknown and highly-expressed basic protein.However,there is room to improve the production level of proteins driven by this promoter.In this thesis,we identified that the region 600 bp to 700 bp upstream of the start codon is critical for the efficiency of the Pcdnal promoter.The β-mannanase production under the control of Pcdna1 was improved by 37.5%by increasing the copy number of this region to three.Screening of several cultivation conditions revealed that the Pcdna1 promoter is heat inducible.Cultivation at 37℃ significantly enhanced the production of β-mannanase and polygalacturonase with the Pcdna1 promoter compared with those at 30℃.Combing the strategies of promoter engineering,multicopy gene insertion,and control of cultivation temperature,the production of β-mannanase could reach 199.85 U/mL in shake flasks,which was 6.6 times higher than that before optimization.Taken together,the results advance the understanding of the widely used promoter Pcdna1 and provide effective strategies for enhancing the production of recombinant proteins in T.reesei.2.Engineering of inducible promoter Pcbh1By engineering Pcbh1,the promoter of T.reesei cellobiohydrolase I encoding gene,specifically including increasing binding sites of transcription activator XYR1 and removing binding sites of transcription repressor CRE1,a highly efficient promoter mutant Pcbh1XdC-1 was obtained.The yield of extracellular β-mannanase driven by Pcbh1XdC-1 was 349.88 U/mL after 144 h fermentation in cellulose medium.Furthermore,Pcbh1XdC-1 was used to drive the expression of β-mannanase while the transcription factor XYR1 mutant was overexpressed.After 96 h fermentation in cellulose medium,the extracellular β-mannanase activity was 447.12 U/mL.In addition,the activity of β-mannanase was 184.11 U/mL after 72 h fermentation in the medium with glucose as the carbon source,which was significantly higher than that of the strain without overexpression of XYR1 mutant.The use of glucose as carbon source in fermentation will be beneficial to further increase the yield of target proteins by fed-batch fermentation.3.Efficient expression of lignocellulose-degrading enzymes based on the engineered transcriptional regulatory elementsThe effective expression of xylanase from Aspergillus niger was achieved by combining strong promoter Pcbh1XdC-1 and overexpressed XYR1 mutant.The xylanase activity of the engineered strain reached 6195 U/mL,22 times that of the original strain PX3,and the protein concentration reached nearly 8 mg/mL.At the same time,the strategy was applied to the expression of endoglucanases from T.reesei.The highest enzyme activity level in cellulose medium was 179.82 U/mL,which was 3.7 times that of the original strain h61.The endoglucanase activity of the engineered strain in the glucose medium could reach 91.33 U/mL,which was 3.3 times that of the original strain h61 under the same condition.The efficient expression of xylanase and endoglucanases further demonstrated that the combination of the strong promoter Pcbh1XdC-1 and the overexpressed XYR1 mutant contributes to efficient protein production in T.reesei.