P27: Identification and deletion of various proteases to increase stability in a Trichoderma reesei host strain

Trichoderma reesei has long been used as a host strain for industrial applications due to its ability to produce large amounts of protein – both native and heterologous. In efforts to increase overall protein yield, Novozymes T. reesei host 1 (NTRH1) underwent several rounds of classical mutagenesis to yield a new host strain, Novozymes T. reesei host 2 (NTRH2).  When NTRH2 was used as a host strain to express heterologous proteins, it was found that some of the proteins were being proteolyzed.  Proteomic analysis was used to identify the top three secreted proteases.  The three proteases identified were a 90 kD subtilisin-like serine protease, a 42 kD aspartic protease, and a 25 kD serine protease.  These proteases were deleted in single, double and triple combinations.  Each strain was then compared with NTRH1 and NTRH2 for increased stability at various temperatures.  Of the single protease deleted strains, it was found that the deletion of the 90 kD subtilisin-like serine protease in NTRH2 was able to show a stability profile much like NTRH1, which has less proteolysis of recombinant proteins.  Deletion of the other two proteases in single deletions did not make a significant difference in stability.  Consequently, any of the double or triple protease deleted strains that contained the 90 kD subtilisn-like serine protease deletion also showed increased stability over the parent strain of NTRH2.  Transcript analysis comparing NTRHI and NTRH2 have shown increased expression of additional proteases, which are now being targeted for deletion in efforts to further improve stability.