Overexpression of a Novel Vacuolar Serine Protease-Encoding Gene (<i>spt1</i>) to Enhance Cellulase Production in <i>Trichoderma Reesei</i>

<i>Trichoderma reesei</i> is widely applied as the major industrial fungus for the production of cellulases used for the conversion of lignocellulosic biomass to biofuels and other biobased products. The protein secretion pathway is vital for cellulase secretion, but few reports are related to the role of the vacuole in cellulase production. Here, we identified a novel vacuolar serine protease gene <i>spt1</i> and investigated the ability of <i>T. reesei</i> to secrete cellulases by disrupting, complementing and overexpressing the <i>spt1</i> gene. Amino acid sequence analysis of the Spt1 protein showed that it belongs to the subtilisin S8 family and has the conserved catalytic triples (Asp, His, Ser) of the serine protease. The deletion of <i>spt1</i> did not lead to a decrease in extracellular protease activity, and the observation of mycelia with the Spt1–eGFP fusion expression and the vacuolar membrane dye FM4-64 staining confirmed that Spt1 was an intracellular protease located in the vacuoles of <i>T. reesei</i>. However, the <i>spt1</i> gene deletion significantly reduced spore production and cellulase secretion, while the <i>spt1</i> complementation recovered these traits to those of the parental strain. When <i>spt1</i> was overexpressed by using its native promoter and introducing multiple copies, the cellulase secretion was improved. Furthermore, a strong promoter, P<i>cdna1</i>, was used to drive the <i>spt1</i> overexpression, and it was found that the cellulase production was significantly enhanced. Specifically, the filter paper activity of the <i>spt1</i> overexpression strain SOD-2 reached 1.36 U/mL, which was 1.72 times higher than that of the parental strain. These findings demonstrated that the <i>spt1</i> gene can be a powerful target for increasing cellulase production in <i>T. reesei</i>, which suggests a possible important role of the vacuole in the cellulase secretion pathway and provides new clues for improving strains for efficient cellulase production.