Cyanamide-inducible expression of homing nuclease I−SceI for selectable marker removal and promoter characterisation in Saccharomyces cerevisiae

Cyanamide-inducible expression of homing nuclease I−SceI for selectable marker removal and promoter characterisation in Saccharomyces cerevisiae

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 r...

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Main Authors: Liam McDonnell, Samuel Evans, Zeyu Lu, Mitch Suchoronczak, Jonah Leighton, Eugene Ordeniza, Blake Ritchie, Nik Valado, Niamh Walsh, James Antoney, Chengqiang Wang, Carlos Horacio Luna-Flores, Colin Scott, Robert Speight, Claudia E. Vickers, Bingyin Peng
Format: Article
Language:English
Published: KeAi Communications Co., Ltd. 2024-12-01
Series:Synthetic and Systems Biotechnology
Subjects:
Metabolic engineering
Genetic circuit
Genome engineering
Synthetic biology
Yeast engineering
Promoter engineering
Online Access:http://www.sciencedirect.com/science/article/pii/S2405805X2400098X
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Summary: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 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 can be developed to facilitate genomic engineering for synthetic biology and metabolic engineering applications.
ISSN:2405-805X

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