Enhanced production of heterologous proteins by the filamentous fungus Trichoderma reesei via disruption of the alkaline serine protease SPW combined with a pH control strategy

• We constructed two plasmids for gene knock-out or expression in Trichoderma reesei.
• We verified the expression of a relatively major extracellular alkaline protease SWP from 20 candidates through qRT-PCR.
• Extracellular protease activity was reduced by deleting the spw gene.
• We observed enhanced production and stability of the heterologous alkaline endoglucanase using the Δspw strain.

The filamentous fungus Trichoderma reesei has received attention as a host for heterologous protein production because of its high secretion capacity and eukaryotic post-translational modifications. However, the heterologous production of proteins in T. reesei is limited by its high expression of proteases. The pH control strategies have been proposed for eliminating acidic, but not alkaline, protease activity. In this study, we verified the expression of a relatively major extracellular alkaline protease (GenBank accession number: EGR49466.1, named spw in this study) from 20 candidates through real-time polymerase chain reaction. The transcriptional level of spw increased about 136 times in response to bovine serum albumin as the sole nitrogen source. Additionally, extracellular protease activity was reduced by deleting the spw gene. Therefore, using this gene expression system, we observed enhanced production and stability of the heterologous alkaline endoglucanase EGV from Humicola insolens using the Δspw strain as compared to the parental strain RUT-C30.