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Cyanamide-inducible expression of homing nuclease I-SceI for iterative genome engineering and parallel promoter characterisation in Saccharomyces cerevisiae

McDonnell, L.; Evans, S.; Lu, Z.; Suchoronczak, M.; Leighton, J.; Ordeniza, E.; Ritchie, B.; Valado, N.; Walsh, N.; Antoney, J. P.; Wang, C.; Scott, C.; Speight, R.; Vickers, C.; Peng, B.

2024-03-27 synthetic biology
10.1101/2024.03.26.586389 bioRxiv
Show 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 {beta}-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.

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"Similar papers" are the closest papers from that journal in the model's embedding space. They show what the match is built on, but the ranking comes mostly from a classifier over the whole training set, not from these examples alone.