A bacterial type-II toxin-antitoxin-mediated gene amplification system in Saccharomyces cerevisiae

Background: 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. Methods: Here, we developed a bacterial type-II toxin-antitoxin-mediated method to manipulate genomic integration of tandem gene repeats in Saccharomyces cerevisiae and further visualised the evolutionary trajectories of gene repeats. We designed a tri-vector system to introduce toxin-antitoxin-driven gene amplification modules. Results: This system delivered 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 gene per copy of genome. Copy-number changes, copy-number increase and copy-number decrease, and stable maintenance were visualised using the green fluorescence protein and blue chromoprotein AeBlue as reporters. Copy-number increases happened spontaneously and independent on a selection pressure. Increased copy number was quickly enriched through toxin-antitoxin-mediated selection. Conclusion: 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.