Universal Expression System for Eukaryotic Microorganisms

This work describes the development of an orthogonal expression system which is functional (transferable) in a large spectrum of eukaryotic microorganisms. The expression system is based on a synthetic transcription factor (sTF), which regulates expression of underlying target gene(s) containing the sTF-dependent promoter(s). The expression of the sTF is achieved by a specific (universal) core promoter, instead of a full promoter, which provides a low, but sufficient expression level of the sTF in a broad spectrum of host organisms. This expression is constitutive, therefore the sTF is present in the cells in all growth conditions, and in all developmental and growth stages. Also the sTF-dependent promoter, regulating the expression of the target gene(s), contains a similar type of universal core promoter functional in diverse species. The combination of the sTF-binding sites and the core promoter in the sTF-dependent promoter enables a wide range of potential target gene expression levels, which can be modulated by the choice of these components from very low to very high. Therefore, the developed expression system provides robust, stable, and tuneable expression levels of the target genes in a broad spectrum of host organisms. This could simplify and focus the genetic tools needed for constructing new expression hosts, including those with undeveloped know-how, and it could enable the use of these (often very promising) hosts in novel industrial applications. The method for selecting the universal core promoters, construction of the expression systems, and demonstration of their performance in diverse hosts, including yeasts and filamentous fungi, will be presented.