Synthetic Toolkit for Complex Genetic Circuit Engineering in Saccharomyces cerevisiae

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.

Model-driven generation of artificial yeast promoters

“…Promoters play a central role in the regulation of protein expression, a key task for both natural and engineered biological systems. In the context of bioengineered systems, precise control of gene expression is critical for tasks such as balancing enzyme expression levels in engineered metabolic pathways [1][2][3] and building gene circuits to control cell behavior based on external stimuli 4,5 . Thus, the availability of large sets of promoters with useful properties has the potential to advance the design of sophisticated genetic constructs.…”

“…The model yeast Saccharomyces cerevisiae is well-characterized and relatively straightforward to genetically engineer 6 ; as such, it is frequently studied in applications spanning microbial biomanufacturing 7,8 , synthetic genomes 9 , and genetic circuit design 5,10 . At present, genetic construct design in this organism generally relies on a small number of well-characterized, native promoters.…”

Tuning up Transcription Factors for Therapy

“…The capacity of LacI and TetR to act as activators or repressors in eukaryotes was compared directly in yeast. The tetR-VP16 AD activated gene expression but the LacI-VP16 failed to do so [81]. When examined whether they can act as a roadblock to repress expression driven by a transcriptional activator, LacI was able to repress transcription, albeit with a lower efficiency than TetR.…”

Tuning up Transcription Factors for Therapy

“…The capacity of lacI and tetR to act as activators or repressors in eukaryotes was compared directly in yeast. The tetR-VP16 AD activated expression but the lacI-VP16 failed to do so [73]. When examined whether they can act as a roadblock to repress expression driven by a transcriptional activator, both lacI and tetR repressed transcription, with tetR displaying a somewhat higher efficiency.…”