We have developed a novel orthogonal expression system that functions in a wide spectrum of eukaryotic microorganisms. The expression system is based upon 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 and thus present in the cells in all growth conditions and in all developmental and growth stages. The sTF-dependent promoter regulating the expression of the target gene also contains a similar type of universal core promoter functional in diverse species. This core promoter can be further modified by inserting synthetic repressor (sR) binding sites, through which an additional expression control is gained upon expression of the sR. The combination of multiple sTFbinding sites and the core promoters (including the repressible versions) 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 tuneable expression levels of the target genes in a broad spectrum of host organisms. Further it simplifies the genetic tools needed for the construction of new protein expression hosts, including those with undeveloped know-how. The method for selecting the universal core promoters, constructions of the expression systems, and demonstrations of their performance in diverse hosts, including yeasts and filamentous fungi, will be presented.
Full conference title:
- Asperfest 14 (2017)