Trichoderma reesei has an extremely high capacity for synthesizing and secreting proteins, thus exhibiting promise as an expression platform for heterologous proteins. However, T. reesei secretes large amounts of native proteins, which hinders its widespread application for heterologous protein production. Here, we designed and built a series of T. reesei chassis using an iterative gene deletion approach based on an efficient genome editing system. Donor DNAs with specially designed construct facilitated screening of positive deletion strains without ectopic insertion. Finally, marker-free T. reesei chassis with lower rates of native protein secretion and low levels of extracellular protease activity were constructed after 11 consecutive rounds of gene deletion. Higher production levels of three heterologous proteins─a bacterial xylanase XYL7, a fungal immunomodulatory protein LZ8, and the human serum albumin HSA─were achieved with these chassis using the cbh1 promoter. It is possible that diverse high-value proteins might be produced at a high yield using this engineered platform.
Keywords: Trichoderma reesei; chassis; heterologous protein expression; iterative gene deletion.