Synthetic Toolkit for Complex Genetic Circuit Engineering in <i>Saccharomyces cerevisiae</i>

TLDR

Sustainable chemical, material, and pharmaceutical production increasingly relies on engineered cell factories, where complex metabolic routes or behavior control systems demand robust, orthogonal gene expression tools. The study develops and characterizes a comprehensive gene expression toolkit that enables accurate, non‑interfering control of gene expression in Saccharomyces cerevisiae. The toolkit consists of synthetic transcription factors functioning as activators or repressors paired with transcription‑factor‑dependent promoters, yielding a broad expression range that exceeds the strongest native promoters. Modularity is shown by a novel bistable circuit that robustly controls a heterologous metabolic pathway and allows on‑demand switching between two alternative metabolic branches.

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.