Genetic Engineering Of Trichoderma Reesei For Heterologous Protein Expression And Improved Cellulase Production

The filamentous fungi Trichoderma reesei can secret a large amount of extracellular cellulases including endoglucanases,cellobiohydrolases,and ?-glucosidases to achieve the efficient degradation of celullose.The high capability of T.reesei in protein synthesis and secretion makes it an excellent host strain for heterologous protein expression,especially for those who require specific posttranslational modification.However,the T.reesei background with a large quantity of endogenous secreted cellulases induced by Avicel partly impeded the isolation and purification of recombinant proteins,while the heterologous expression driven by the commonly used strong cbhl promoter is dependent on cellulase-induction conditions.Although the cellulases produced by T.reesei have been widely applied in industry,its cost is still a main limiting factor for the production of biofuels such as ethanol.Therefore,the improvement of cellulase production by T.reesei industrial strain is essential for social and economic development.In the present study,we performed strain engineering for T.reesei at the molecular level,and the main work can be summarized in the following two parts:Firstly,deletion of several main cellulase genes and overexpression of the transcriptional activator Xyrl led to a significant decrease in the background of endogenous secreted proteins,and the inducing carbon source-independent expression of proteins under the control of cbhl promoter,both of which largely facilitated the expression and purification of heterologous proteins.Secondly,the overexpression of the transcriptional activator Xyrl or the ?-glucosidase Bgll remarkably enhanced the extracellular cellulase production.The main results were presented as follows:1.Two T.reesei strains,A5a7b6aOExyr1 and A5a7b6a7aOExyr1 were successfully constructed,wherein the main cellulases Ce15a,Ce17b,Ce16a,or Ce17a were absent and the transcriptional activator Xyrl was overexpressed.These two strains are the ideal host strains for heterologous protein expression under the control of cbhl promoter,as demonstrated by their significantly low background of endogenous protein expression and induction-independent expression of target genes even on cellulase-repressing carbon sources.The ?5a7b6a strain was derived from QM9414 in which cel5a/cel7b encoding endoglucanase ?/? and cel6a encoding cellobiohydrolase ? were first deleted by homologous recombination.SDS-PAGE analysis demonstrated that extracelluar proteins of this strain were decreased significantly.Xyr1 was then overexpressed in ?5a7b6a to obtain the ?5a7b6aOExyr1 strain.The produced CbhI by this strain under glucose was as much as or even more than that of the wild-type strain on Avicel.It thus indicated that protein expression under cbh1 promoter in this strain could be achieved on either glucose or Avicel.cbhl was further knocked out in the ?5a7b6aOExyr1 strain to acquire the ?5a7b6a7aOExyr1 strain,in which the random integration of the target gene expression cassette could be implemented.Although this strain could not utilize Avicel,protein expression under the cbhl promoter was allowed on glucose,and deletion of cbhl reduced the background of secreted enzymes further and greatly facilitated protein purification.2.Endoglucanase NCE5 from Humicolo insolens was successfully expressed in?5a7b6aOExyr1.Zymogram analysis showed that the optimum pH of this enzyme was 6NCE5 is an endoglucanase derived from H.insolens with a theoretical molecular weight of 23 kDa.The coding sequence of NCE5 was optimized according to the codon usage frequency of T.reesei,and its expression cassette under cbhl promoter was constructed.The cassette was targeted into the cbhl locus in ?5a7b6aOExyr1 strain.Zymogram analysis showed that NCE5 was successfully expressed in ?5a7b6aOExyr1 with an optimum pH of around 6.3.Xyrl and ?-glucosidase Bgll were overexpressed in cellulases high-yielding industrial T.reesei strain C10,respectively.The filter paper activity of their recombinant strain increased by 25%and 30%C10 is a cellulase hyper-producing T.reesei strain.The C10-OExyrl strain was obtained by expressing xyr1 under the tcu-1(encoding a copper transporter)promoter.The engineered T.reesei showed 25%higher filter paper activity and earlier protein secretion time compared with the parent strain.C10-bgll strain was obtained by overexpressing the ?-glucosidase encoding gene bgll under the cbhl promoter and the filter paper activity of the recombinant strain was improved by about 30%.