The integration of tandem gene repeats via a bacterial type-II toxin-antitoxin-mediated gene amplification (ToxAmp) system and stability visualisation in Saccharomyces cerevisiae

Tandem gene repeats naturally occur as important genomic features and determine many traits in living organisms, like human diseases and microbial productivities of target bioproducts. Here, we develop a bacterial type-II toxin-antitoxin-mediated method to manipulate genomic integration of tandem gene repeats in Saccharomyces cerevisiae and further visualise the evolutionary trajectories of gene repeats. We designed a tri-vector system to introduce toxin-antitoxin-driven gene amplification (ToxAmp) modules, and accidentally re-visited the high-level capacity of multi-fragment co-transformation in S. cerevisiae. This system delivered the multi-copy gene integration in the form of tandem gene repeats spontaneously and independently from toxin-antitoxin-mediated selection. Inducing the toxin (RelE) expressing via a copper (II)-inducible CUP1 promoter successfully drove the in-situ gene amplification of the antitoxin (RelB) module, resulting in ~40 copies of a green fluorescence reporter (GFP) gene per copy of genome. The copy-number changes, increasing and decreasing, and stable maintenance were visualised using the GFP and blue chromoprotein AeBlue as reporters. Copy-number increasing happened spontaneously not depending on a selection pressure and was quickly enriched through toxin-antitoxin-mediated selection. In summary, the bacterial toxin-antitoxin systems provide a flexible mechanism to manipulate gene copy number in eukaryotic cells and can be exploited for synthetic biology and metabolic engineering applications. ### Competing Interest Statement Laura Navone declares competing interests in Eden Brew (Australia). Others declare no competing interests.