Cyanamide-inducible expression of homing nuclease I- SceI for selectable marker removal and promoter characterisation in Saccharomyces cerevisiae.
Synth Syst Biotechnol
; 9(4): 820-827, 2024 Dec.
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| MEDLINE
| ID: mdl-39072146
ABSTRACT
In synthetic biology, microbial chassis 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 expression, which triggered double-strand DNA breaks (DSBs) for selectable marker removal. We further combined cyanamide-induced I- SceI-mediated DSB and maltose-induced MazF-mediated negative selection for plasmid-free in situ promoter substitution, which simplified the molecular cloning procedure for promoter characterisation. We then characterised three tetracycline-inducible promoters showing differential strength, a non-leaky ß-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 can be developed to facilitate genomic engineering for synthetic biology and metabolic engineering applications.
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Synth Syst Biotechnol
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2024
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Article