Construction A Protease Deficient Strain Trichoderma Reesei RUT-C30by Genetical Modification

Trichoderma reesei is one kind of filamentous fungi which has been widely applied in the industrial production.In the past, people used UV mutagenesis rather than genetic engineering to transfom T. reesei in order to raise the cellulose activities. Some of transformation strains achieved good results such as the RUT-C30strain. Since the whole-genome sequencing of T. reesei had been completed and a new method named as Agrobacterium tumefaciens-mediated transformation had been created, we could transform the T. reesei more accurately and conveniently. There were many attempts on expressing heterologous proteins in T. reesei, but the results were not satisfactory. After analyzing this phenomenon and combining with some reports in literatures, we found the bottleneck may be caused by the self-produced protease. Through reviewing literature and aligning sequences, we found three kinds of proteases:serine protease, aspartic protease vacuolar protease in T. reesei RUT-C30. After we analyzed the signal peptide sequences and RT-PCR experiments, we chose to knock out the serine protease as the first step to transform T. reesei RUT-C30strain.First, we constructed a universal plasmid to make the further experiments faster and more effective. Then we transformed the selection marker of T. reesei reducing the application restriction. We knockouted the serine protease gene in T. reesei successfully after the adequate pre-experiments. As a result, the extracellular protease decreased by50%and the endo-cellulase activity of T. reesei increased by25%to30%.Also T. reesei is a good gene pool, while protease is the most important enzymes in industrial enzyme production. We tried to express the serine protease of T. reesei heterologously. When use the Pichia pastoris as a host, we got a prtential enzyme activity as53U/mL.