An orthogonal transcription system for Saccharomyces cerevisiae

Metabolic engineering of microbial production hosts with long, heterologous pathways strongly increases the burden on a production strain. Thus, genomic stability and robust expression tools (decoupled from the host metabolism and regulation) are key for the creation of robust production strains. Here we describe the development of an orthogonal transcription system using short promoters for Saccharomyces cerevisiae. This system is based on an endogenous transcription system found in baker's yeast and is similar to the bacteriophage T7 expression system for E. coli. An additional copy of the polymerase coding sequence and the one of its transcription factor were introduced into the genome of S. cerevisiae, together with the mOrange coding sequence. The latter was placed under the control of such a short promoter. The absence of mOrange fluorescence in cells expressing the mOrange gene under control of this short promoter, with and without expression of the polymerase proved the orthogonality of the transcription system. Further characterization of the promoter structure, fine tuning of the transcription factor expression and the construction of promoter libraries will enable the full exploitation of this tool in yeast synthetic biology approaches. Finally, it will increase the genomic stability and performance robustness of industrial production strains