Construction Of Binary Vectors And Neutral Cellulase Expression In Trichoderma Reesei

Filamentous fungus Trichoderma reesei is an extremely efficient producer of cellulase. The cellulase system of T. reesei is composed of several enzymes that function in a complex. This complex consists of endoglucanases (EG), cellobiohydrolases (CBH), and cellobiases (CB). Genetic engineering has been used to tailor cellulase properties and increase its production. Several mutant strains have been engineered to produce cellulases at approximately 40 g/L, and the major cellulase, cellobiohydrolase I (CBHI), accounts for approximately 50% of all secreted proteins.Thus, the cbhl promoter has been considered one of the strongest promoters in T.reesei. Additionally, T. reesei itself is not toxic. Even under engineered conditions of maximal enzyme production, T. reesei does not produce mycotoxins or antibiotics. Therefore, it is ideal to use the cbhl promoter and terminator to construct expression vectors in order to produce homologous and heterologous proteins in T. reesei.We conducted the present study to construct two binary fungal expression vectors, both with the strong fungal promoter of cbhl. One vector, pWEF31, can be used for random integration, and the other, pWEF32, allows for site-directed integration. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase1 gene (cbhl) from T. reesei. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30.In our lab, we used pWEF31 to insert a fluorescent protein reporter into the T. reesei genome in order to analyze inducible expression of an enzyme-reporter. We can also use pWEF31 to insert two neutral cellulase into the T. reesei genome, resulting in optimal pH for cellulase activity, which occurs between 4.8 and 6.0.