A light-switchable bidirectional expression system in filamentous fungus Trichoderma reesei

- A “light-on” bidirectional promoter was firstly constructed in T. reesei.
- A “light-off” promoter was constructed in T. reesei for the first time.
- A native cellulase-free background for the expression was achieved in T. reesei.

The filamentous fungi Trichoderma reesei is widely used in the production of cellulolytic enzymes and recombinant proteins. However, only moderate success has been achieved in expressing heterologous proteins in T. reesei. Light-dependent control of DNA transcription, and protein expression have been demonstrated in bacteria, fungi, and mammalian cells. In this study, light inducible transactivators, a “light-on” bidirectional promoter and a “light-off” promoter were constructed successfully in T. reesei for the first time. Our light inducible transactivators can homodimerize and bind to the upstream region of artificial promoters to activate or repress genes transcription. Additionally, we upgraded the light-inducible system to on-off system that can simultaneously control the expression of multiple heterologous proteins in T. reesei. Moreover, a native cellulase-free background for the expression of heterologous proteins was achieved by knocking out the genes involved in transcriptional regulation and encoding of cellulases: xyr1, cbh1, and cbh2. Our light-switchable system showed a very little background protein expression and robust activation in the blue light with significantly improved heterologous protein expression. We demonstrate that our light-switchable system has a potential application as an on/off “switch” that can simultaneously regulate the expression of multiple genes in T. reesei under native cellulase-free background.