Protease regulatory factors of Trichoderma reesei can be controlled to improve therapeutic protein production

Christopher Landowski 1 Ann Westerholm-Parvinen 1 Bernhard Helk 2 Juhani Saarinen 3 Markku Saloheimo 1
1Protein production, VTT Technical Research Centre of Finland Ltd., Espoo, Finland
2Novartis, Pharma AG., Basel, Switzerland
3Glykos, Finland Ltd., Helsinki, Finland

Protease secretion limits the production of many sensitive therapeutic proteins such as hormones and cytokines that are by nature easy to degrade. There are over 40 potential proteases secreted by Trichoderma reesei. We looked for transcriptional regulators of these proteases with the aim to control and reduce the expression of a wide range of proteases. Protease induction studies were set up to trigger protease activity with peptide and protein substrates in liquid cultures of T. reesei. Genome-wide expression data was generated and clustered to find out what genes are co-regulated after different treatments. Twelve candidate transcription factors or regulatory proteins were selected as potentially being involved in upregulating protease activity. To narrow the selection, the regulator genes were located on the scaffold to see if they were physically near any protease genes. Transiently silencing ptf1, prp1, and ptf3 with siRNA downregulated the expression of a selection of protease genes in accordance with the co-regulation observed. Treatment with both ptf1 and prp1 siRNAs increased the effectiveness of the knockdown and reduced protease activity. The deletion of single, double, and triple combinations of the regulators successfully reduced protease activity and increased interferon alpha 2b production. For example, the triple deletion Δptf1Δprp1Δptf8 lead to a 3.7-fold improvement in interferon alpha 2b production. In conclusion, we have demonstrated that silencing or deleting protease regulatory factors can broadly reduce protease activity.