Controlling the expression of the Trichoderma reesei subtilisin protease gene slp2 leads to improved biotherapeutic protein production

T. reesei is capable of high levels of protein production, but is also an active secretor of proteases. This hinders the production of many sensitive therapeutic hormones and cytokines that are by nature easy to degrade. Even antibodies which are thought to be relatively stable molecules are susceptible to protease degradation. The subtilisin like protease SLP2 is one of the most abundant proteases produced by T. reesei and causes degradation of a range of therapeutic proteins such as antibodies and interferon alpha 2b. To control the expression of slp2 gene we tried to delete it, but that strategy affected the growth and sporulation of the strain. Alternatively we developed RNA hairpin expressing vectors to silence its messenger RNA or we simply exchanged the promoter of slp2 to attempt to reduce its expression. In a mAb production strain these approaches reduced the secreted protease activity by half and improved the antibody yield by 2-fold, with the highest production level reaching 7.6 g/L. The heavy chain quality was strikingly improved after controlling the SLP2 protease activity. Likewise, silencing slp2 also dramatically improved the production of interferon alpha 2b, allowing levels of 7.9 g/L to be reached.