Cyanamide-inducible Expression of Homing Nuclease I for Selectable Marker Removal and Promoter Characterisation in

In synthetic biology, microbial chassis including yeast 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 () promoter in . Here, we prototyped the cyanamide-induced I expression, which triggered double-strand DNA breaks (DSBs) for selectable marker removal. We further combined cyanamide-induced I-mediated DSB and maltose-induced MazF-mediated negative selection for plasmid-free promoter substitution, which simplified the molecular cloning procedure for promoter characterisation. We then characterised three tetracycline-inducible promoters showing differential strength, a non-leaky β-estradiol-inducible promoter, cyanamide-inducible promoter, bidirectional promoters, and five pairs of bidirectional promoters. Overall, alternative regulatory controls for genome engineering tools can be developed to facilitate genomic engineering for synthetic biology and metabolic engineering applications.