Cyanamide-Inducible Expression of Homing Nuclease I-Scei for Iterative Genome Engineering and Parallel Promoter Characterisation in Saccharomyces Cerevisiae

Abstract

In synthetic biology, microbial chasses including yeast Saccharomyces cerevisiae are iteratively engineered with increasing complexity and scale. Wet-lab genetic engineering tools are developed and optimised to facilitate strain construction but are often incompatible with each other due to shared regulatory elements, such as the galactose-inducible (GAL) promoter in S. cerevisiae. Here, we prototyped the cyanamide-induced I-SceI-mediated double-strand DNA breaks (DSBs) for selectable marker recycling in yeast metabolic engineering. We further combined cyanamide-induced I-SceI-mediated DSB and maltose-induced MazF-mediated negative selection for plasmid-free in situ promoter replacement, which simplified the molecular cloning procedure for promoter characterisation in S. cerevisiae. We then characterised three tetracycline-inducible promoters of differential strength, a non-leaky β-estradiol-inducible promoter, cyanamide-inducible DDI2 promoter, bidirectional MAL32/MAL31 promoters, and five pairs of bidirectional GAL1/GAL10 promoters. Overall, alternative regulatory controls for genome engineering tools are important for the construction of complexed genotypes in microbial systems for synthetic biology and metabolic engineering applications.

Keywords: Metabolic engineering, genetic circuit, genome engineering, synthetic biology, yeast engineering, promoter engineering