| Lignocellulose is the most abundant renewable bioresources in the world.It can be converted into biofuels,bio-based chemicals and bio-based materials through biorefining.The bioconversion of lignocellulose has attracted widespread attention,because of its environmental friendliness,low energy consumption and mild reaction conditions.One of the bottlenecks of the bioconversion of lignocellulose is the high cost of cellulase,and how to reduce the cost of cellulase is one of the current research hotspots.Commercial cellulase preparations are mainly produced from fungi,such as Trichoderma reesei.Trichoderma orientalis EU7-22 isolated and mutated by our lab has full of components of cellulase system and hemicellulase system.In the previous research,the main cellulase,hemicellulase and some transcriptional regulatory genes of Trichoderma orientalis EU7-22 have been cloned,and the regulatory factors of Cre1 and Ace3 on cellulase production of Trichoderma orientalis EU7-22 have been studied.The further study on the cellulase regulatory could elucidate the complex cellulase regulation system and improve its industrial application.In this thesis,the effects of regulators Cre2,Acel and Xyr1 on cellulase production of Trichoderma orientalis EU7-22 were detailed studied.The gene of xylanase 3 and its flanking region were cloned,and the engineered strains with high ?-glucosidase activity and high cellulase activity were constructed.The main results of this research are as follows:(1)The gene and its 5 'and 3' flanking sequences of Trichoderma orientalis EU7-22 were cloned by degenerate PCR and hi TAIL PCR.The cre2 gene including 3 introns was 2516 bp,and the 5' and 3' flanking sequences were 4409 bp and 1300 bp,respectively.The Cre2 protein is 741 aa,and has 95%homologous with the Creb of Trichoderma reesei QM6a.The cre2 gene of Trichoderma orientalis was disrupted by homologous recombination,and the colony morphology and spore formation of EU7-22 and Acre23 were almost the same when they were grown on the medium with different carbon source.The cellulase activity and protein concentration of ?cre2-3 strain were earlier up to the highest activity than parental strain EU7-22 under inducing condition and the FPase and PNPGase activities were higher than that of parental strain.Although hemicellulase activity of ?cre2-3 was earlier up to the highest than parental strain EU722,the xylanase activity and PNPXase activity of ?cre2-3 was obvious reduction relative to EU7-22.The maximum activities of the xyanase and ?-glycosidase of?cre2-3 were 33.53%and 81.89%lower than that of EU7-22.This results have not been reported,it stated that the Cre2 regulating the celluase and hemicellulas production in Trichoderma orientalis has a certain strain specificity.(2)The 939 bp 5' flank sequence and 1626 bp 3' flank sequence of the ace1 of Trichoderma orientalis EU7-22 were cloned by hi TAIL PCR,then the strain disrupted the ace1 gene was constructed.The phenotype of ?ace1-1 was almost the same as that of the parental strain EU7-22 on different carbon source plates.The growth rates of?ace1-1 on the plates were lower than parental strain EU7-22 on different carbon resource plates expect the glucose,on which it was slightly faster than EU7-22.The main cellulase and hemicellulase activities of ?ace1-1 strain were earlier up to the highest than that of parental strain EU7-22.The highest activities of cellulase and hemicellulase of ?ace1-l were higher than that of parental strain EU7-22.The activities of FPase,CMCase and xylanase in ?ace1-1 were 114%,103%and 118%of that of EU7-22.In order to analysis the synergistic effect of regulators Acel and Cre2 on cellulase production in Trichoderma orientalis EU7-22,the ?ace1 cre2-5 strain was constructed by cre2 knockout cassette using G418 as screening marker based on the ?ace1-1 strian.?ace1 cre2-5 strain grew faster than the parental strain EU7-22 on plate with glucose as carbon source,but it grew significantly slower than EU7-22 on the plate with cellulose or hemicellulose as carbon sources.Although the major cellulase and hemicellulase activities of ?ace1 cre2-5 were earlier up to the highest during induction culture,all the cellulase and hemicellulase activity decreased relative to the paraental strain.Compared with the parental strain EU7-22,the activities of FPase,CMCase,PNPCase,PNPGase,xylanase,and PNPXase in ?ace1 cre2-5 were reduced by 14%,16%,32%,30%,10%and 49%,respectively.This result indicate that the absence of the two negative regulators both reduced the cellulase production of Trichoderma orientalis rather than improved the cellulase production.(3)The 3164 bp 5' flanking sequence and 1379 bp 3' flanking sequence of xyr1 from Trichoderma orientalis EU7-22 were cloned by hi TAIL PCR.Based on the flanking regions,the ?xyr1-3 disrupted the xyr1 gene was constructed.The ?xyr1-3 strain could hardly detect any cellulase and hemicellulase activity in inducing culture,indicating that transcription regulator xyr1 is a key activator for cellulase and hemicellulase expression in Trichoderma orientalis EU7-22.xyr1 was constitutive overexpressed in Trichoderma orientalis EU7-22 by the promoter and terminator of pyruvate decarboxylase gene from Trichoderma reesei QM9414,and the cellulase activities of the 5 transformants were significantly increased compared with that of the parental strain,among which the cellulase activity of strain dxyr-1 was the highest.The activities of FPase,CMCase,PNPCase and xylanase of dxyr-1 strain were 183%,198%,200%and 110%of those of the parental strain EU722,respectively.However,the PNPGase activity of dxyr1-1 did not increase,and the PNPXase activity even decreased significantly compared with the EU7-22.In general,over-expression of xyr1 is an effective ways to increase the cellulase activity of Trichoderma orientalis.(4)The xyn3 gene of Trichoderma orientalis EU7-22 and its flanking sequence were cloned by degenerated PCR and chromosome walking.The xyn3 with three introns was 1283 bp,the 5' and 3' flanking region were 3904 bp and 1476 bp,respectively.The XYN? proetien has 348 aa and the 1-14 aa is the predicted signal peptides.The molecular weight of the mature protein is 36.55 KDa,and its isoelectrical point is 6.14.According to the analysis of phylogenetic tree,the XYN? belongs to the glycoside hydrolase 10 family,and is mostly homologous to Trichoderma pseudokoningii endoxylanase.The ?-glycosidase 1 was overexpressed in Trichoderma orientalis EU7-22 using the promoter and terminator of xyn3.The PNPGase activities of the transformants bgl1 and bgl-2 were 150%and 300%higher than the parental strain EU7-22,and the FPase activities of them were 17%and 35%higher.These results demonstrate that it is possible to construct high production of ?-glycosidase in Trichoderma orientalis using the promoter of xyn3.5)The transcript activator Aces was constitutive overexpressed in recombinant strain dxyr-1 using the promoter and terminator of enolase from Trichoderma reesei QM6a.Eight transformants were obtained,among which 6 strains showed enhanced cellulase activity,and the highest cellulase activity strain was dxyA-8.After 4 days'culture under inducing,the activities of FPase,CMCase,PNPCase,PNPGase,xylanase and protein concentration of dxyA-8 were 334%,223%,264%,168%,165%and 206%of those of the parental strain EU7-22,respectively.After 4 days' culture in glucose medium,the activities of FPase,CMCase,PNPCase,PNPGase,xylanase and protein concentration of dxyA-8 were 3163%,4719%,3939%,233%,616%and 253%of those of the parental strain EU7-22,respectively.The FPase and CMCase activities of dxyA8 were higher than that of parental strain under inducing condition,up to 2.55 IU/mL and 90.38 IU/mL,respectively.Reducing sugars in the hydrolysate released by cellulase of dxyA-8 increased about 24%compared with that of EU7-22 when the equal volume of crude enzyme produced by EU7-22 and dxyA-8 under inducing condition for 4 days was used for hydrolyzing NaOH/H2O2 pretreated Spartina.In summary,this dissertation studied the effects of regulators Cre2,Acel and Xyrl on the production of cellulase and hemicellulase in Trichoderma orientalis EU7-22,and also analyzed the synergistic effects of Cre2 and Ace1 on the production of cellulase and cellulase.The xyn3 gene and its flanking sequence of Trichoderma orientalis EU722 were cloned,then the promoter and termination of xyn3 were used to overexpress bgl1 in Trichoderma orientalis EU7-22 and the strain of hyper-production of glucosidase was construted.At last,high-producing cellulase strain was obtained by constitutive overexpression both xyr1 and ace3.These results were meaningful for the construction cellulase hyper-production strain using Trichoderma orirentalis EU7-22. |
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