SES: Universal expression system for fungi

Anssi Rantasalo Christopher P. Landowski Joosu Kuivanen Jussi Jäntti Dominik Mojzita
Industrial Biotechnology, VTT Technical Research Centre of Finland Ltd., Espoo, Finland

We have developed a novel orthogonal expression system (SES) that functions in a wide spectrum of eukaryotic organisms. The expression system is based on a synthetic transcription factor (sTF) that regulates expression of the target gene via a sTF-dependent promoter. The sTF expression is driven by an engineered, universal core promoter that provides a low, but sufficient expression level of the sTF. The sTF expression is constitutive in all tested growth conditions and developmental and growth stages. The sTF-dependent promoter regulating the expression of the target gene also contains a similar type of universal core promoter, making the whole expression system independent of the host’s native regulation and therefore functional in diverse species. The combination of multiple sTF-binding sites and the core promoters enables specific adjustment over a wide range of target gene expression levels, from very low to very high. This expression system provides robust, stable, and tunable expression levels of target genes in a broad spectrum of host organisms with numerous applications in metabolic engineering and protein/enzyme production. Furthermore, it greatly simplifies the genetic tools needed for the construction of novel production hosts, including those with undeveloped know-how for the heterologous gene expression.

The method for selecting the universal core promoters, constructing the expression systems, and demonstrating their performance in diverse fungal species will be presented. In addition, the utility of the expression system will be demonstrated on heterologous gene expression in novel fungal hosts, which were previously not genetically modified.