Construction Of Chimeric Transcription Factors And Combinatorial Genetic Engineering Of Strains For High Cellulase Production In Penicillium Oxalicum

Lignocellulosic biomass can be degraded by cellulase,and the intermediates and final products produced by the degradation can replace the fuel and other chemicals produced from fossil raw materials to some extent.At present,the high cost of cellulase limits the widespread use of lignocellulose.Therefore,it is of great value and significance to increase cellulase production and reduce cellulase production costs.The expression of cellulase genes in filamentous fungi is mainly regulated at the transcriptional level.Studies have shown that multiple transcriptional regulators(positive regulators such as ClrB and XlnR,as well as negative regulators such as CreA and ACEI)are involved.Construction of transcription factors with enhanced or constitutive activating ability on cellulase expression is expected to significantly improve the production level of cellulases.Based on two key cellulase transcriptional regulators(ClrB and CreA)in Penicillium oxalicum,we constructed some artificial chimeric transcription factors that promote the transcription of cellulase genes,and combined with other engineering targets to construct high cellulase-producing strains.The main research contents and results of this thesis are as follows:1.Construction and functional analysis of artificial transcription activators based on the DNA-binding domain of ClrBBioinformatics method was used to predict the DNA binding domain of ClrB and other transcription factors.The DNA binding domain sequence(clrBBD)of ClrB was fused with the sequences apart from DNA binding domains of AmyR,XlnR and XlnRA871V to construct chimeric transcription factors CA,CX,CXC.The clrBBD sequence was also fused with the transcriptional activation domain sequence of Gal4 to construct a chimeric transcription factor CG,and assembled in tandem with the C-terminal sequence of xlnRA871V and VP64 to construct the chimeric transcription factor CXCV.In addition,using the promoter of bgl2 to drive the expression of the CXc-encoding gene,a gene expression cassette bCXc with positive feedback activity in theoretical was constructed.The cellulase activities of the above chimeric transcription factor expression strains under different culture conditions were determined.It was found that the gCXc strategy(expressing CXC using gpdA promoter)had the best effect on cellulase activity in the inducing medium.Under the repressing condition with glucose as a carbon source,no strain showed obvious cellulase synthesis.2.Engineering of the functional domain of carbon catabolite repression transcription factor CreACreA is a key repressor protein that inhibits the expression of cellulase in P.oxalicum.We designed a chimeric transcription factor CrCl in which the DNA binding domain of CreA and the transcriptional activation domain of ClrB were fused together.In addition,we added the upstream and downstream sequences of creA at the ends of the gene sequence of CrCl,which was used to knock out the original creA while expressing CrCl.This chimeric transcription factor was expected to remove the inhibitory effect of CreA on cellulase expression,while at the same time exerting the activation of cellulase expression through the ClrB transcriptional activation domain.Under cellulose-inducing condition,filter paper activity of the resulted CrCl-?creA strain was 1.75 times that of the original strain,but lower than the creA-deleted strain.Under glucose condition,the cellobiohydrolase activity and cellobiohydrolase gene transcript abundance of CrCl-?creA were higher than that of creA-deleted strain.3.Combinatorial genetic modification of cellulase genes and regulator genesBased on the comparison of chimeric transcription factors,other cellulase transcriptional regulators and multiple cellulase genes were genetically engineered to further increase cellulase production and optimize cellulase mixture composition.Firstly,the M12-gCXC strain with the highest cellulase activity was selected,in which the gene bgl2 encoding an intracellular ?-glucosidase was replaced by an extracellular P-glucosidase gene bgll derived from Aspergillus niger,resulting the strain gCXC-bB.In complex inducing medium,extracellular ?-glucosidase activity increased by 495 times than that of M12-gCXC.Further,the carbon catabolite repression transcription factor creA was knocked out and the gene Trcbh1 encoding the cellobiohydrolase from T.reesei was expressed,generating the strain gCXC-bB-cC.Fermentation under complex inducing condition showed that the filter paper activity of the gCXc-bB-cC strain was 4.2 times that of the M12-gCXC strain,reaching 14.4 U/ml,and the cellobiohydrolase activity was 8.7 times that of the M12-gCXC at 96 h.