| T.reesei is widely used in industry and as a vector for the expression of heterologous protein due to its large ammount of secreted protein(100 g/L),genetic stability,rapid growth rate,simple cultivation and safety.In addition,T.reesei can secrete a kind of yellow pigment named as sorbicillin with the character of anti-cancer,anti-viral and anti-bacteria.Otherwise,the accomplishment of the genome sequencing of several strains of T.reesei provides a solid foundation for construction of gene engineering strains with good properties and studying the cellulase production mechanism.In this paper,three gene engineering strains TRB1,SEU-7 and ZC121121 were obtained.TRB1 produced more cellulases than RUT-C30 and released CCR effect,SEU-7 produced the most FPase to this day on lactose.ZC 121121 could produce abundant yellow pigments.Then DsRed was used to label the major cellulases and study the secretion mechanism of the three major cellulases.Finally,we further investigated the cellulase and sorbicillin production mechanism by RNA-seq and qRT-PCR.The major contributions are as follows:1.A ?-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role and mechanism of CEL3D in cellulase productionThe T.reesei recombinant strain TRB1 was constructed from T.reesei RUT-C30 by the T-DNA-based mutagenesis.qRT-PCR and gene cloning showed that in TRB1 ?-glucosidase CEL3D was mutated through the random insertion by AMT and was not expressed.2.Cellulase hyper-production by Trichoderma reesei mutant SEU-7 on lactoseT.reesei mutant strain SEU-7 was constructed from T.reesei RUT-C30 with the overexpression of endogenous gene cel3a(bgll)by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation(AMT).Compared to RUT-C30,SEU-7 displays substantially enhanced activities of both cellulase and hemicellulase when grown on either lactose or cellulose.The induction efficiency with lactose was found to be higher than cellulose in strain SEU-7.To the best of our knowledge,we achieved the highest FPase activity in SEU-7 in both batch culture(13.0 IU/mL)and fed-batch culture(47.0 IU/mL)on lactose.Moreover,SEU-7 displayed unrivaled pNPGase activity on lactose in both batch-culture(81.0 IU/mL)and fed-batch culture(144.0 IU/mL)as compared to the other reported T.reesei strains in the literature grown in batch or fed-batch experiments on cellulose or lactose.Afteterwards,qRT-PCR,qPCR and Genome sequencing were performed to study the mechanism for cellulase hyper-production on different carbon sources.3.Collateral mutation produced in the process of deletion of gene 121121 switched the cellulase production to the yellow pigment production pathway in Trichoderma reeseiFor the first time,Collateral mutation produced in the process of deletion of gene 121121 was proved to be a transcriptional regulator for the convertion between cellulase production and the yellow pigment(sorbicillin)production.Collateral mutation produced in the process of deletion of gene 121121 reduced cellulase production but increased the production of sorbicillin.We performed RNA-seq for more informations of ZC121121,then HPLC-MS was used to identify the metabolites of ZC 121121 and OE121121.4.Tracking the production,distribution and secretion of the main cellulases in Trichoderma reeseiIn our study,we successfully engineered several transformants overexpressed the cel7b,cell a and cel3a gene labeled with DsRed as a tracking gene,designated as RCMC,RCBH and RBGL,through the AMT method.Results showed that BGL1 appeared earlier than the other two cellulases in vivo,however the secretion of BGL1 was slower than the other two cellulases.Microscopy observation showed that BGL1 and CBH can be secreted onto cell memebrane,but not for CBH.Membrane staining with cholesterol-PEG-FITC reinforced the above result.Moreover,we proved that all three cellulases can be entered into ER for processing by co-staining with ER-Green and BGL1 and CMC secreted through Golgi but not or less for CBH by co-staining with GolgiGreen. |
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