Abstract
Sustainable production of chemicals, materials, and pharmaceuticals is increasingly performed by genetically engineered cell factories. Engineering of complex metabolic routes or cell behavior control systems requires robust and predictable gene expression tools. In this challenging task, orthogonality is a fundamental prerequisite for such tools. In this study, we developed and characterized in depth a comprehensive gene expression toolkit that allows accurate control of gene expression in Saccharomyces cerevisiae without marked interference with native cellular regulation. The toolkit comprises a set of transcription factors, designed to function as synthetic activators or repressors, and transcription-factor-dependent promoters, which together provide a broad expression range surpassing, at high end, the strongest native promoters. Modularity of the developed tools is demonstrated by establishing a novel bistable genetic circuit with robust performance to control a heterologous metabolic pathway and enabling on-demand switching between two alternative metabolic branches.