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.

Camptothecin and its derivatives are the third largest anticancer drugs in the world market, mainly used to treat malignant tumors such as lung, colon and cervical cancer. Camptothecin was firstly discovered in Camptotheca acuminate and extracted mainly from C. acuminate and Nothapodytes nimmoniana for medicine production (Sadre et al. 2016). However, the overharvesting of C. acuminate and N. nimmoniana has greatly reduced their populations in nature, which are currently listed as the second protected plants in China and India. It is estimated there would be 20 million new cancer cases in 2025 all over the world, meeting the growing demand for camptothecin and other anti-cancer drugs has become a daunting challenge (Seca et al. 2018). In this study we tried to elucidate the unknown biosynthetic pathway from strictosamide 1 to strictosamide epoxide 2 by unearthing the candidate enzymes from the proteome of plant Ophiorrhiza pumila using the chemoproteomic strategy.