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.
Highlights
Sustainable production of chemicals, materials, and pharmaceuticals is increasingly performed by genetically engineered cell factories
The gene expression tools are based on the use of synthetic transcription factors consisting of modular parts, such as DNA binding proteins, receptor domains, and transcription activation domains.[3,5−9] Modularity of the different parts is a great advantage as it allows design of sTFs with desired functions and target specificities
The existing parts typically originate from evolutionarily distant organisms, such as bacterial repressor proteins,[6,9−11] zinc finger domains,[3,5,8,11] transcription activator-like effectors (TALEs),[12] or the Cas[9] null mutant protein,[12−14] which have no homologues in yeast and potentially provide a high degree of orthogonality
Summary
Sustainable production of chemicals, materials, and pharmaceuticals is increasingly performed by genetically engineered cell factories. The use of homologous (native) DNA regulatory parts in an expression system can lead to unintended behavior that is caused, for instance, by changing growth conditions during extended bioprocesses This is due to the often poorly understood, complex native cellular regulation in which several transcription factors and other regulators are involved and can influence the performance of the expression system.[3,4] Adjustability, expression output stability and predictability, the ability to reach high expression levels, and independence on specific inducer compounds are desirable features of gene expression tools. The values represent the mean of two biological and two technical replicates ± SD
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