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

Evans, S.; Lu, Z.; McDonnell, L.; Anderson, W.; Peralta, F.; Watkins, T.; Ahmed, H.; Luna-Flores, C. H.; Loan, T. D.; Navone, L.; Trau, M.; Scott, C.; Speight, R.; Vickers, C.; Peng, B.

doi:10.1101/2024.01.30.578080

bioRxiv · 10.1101/2024.01.30.578080

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

Evans, S.·Lu, Z.·McDonnell, L.·Anderson, W.·Peralta, F.·Watkins, T.·Ahmed, H.·Luna-Flores, C. H.·Loan, T. D.·Navone, L.·Trau, M.·Scott, C.·Speight, R.·Vickers, C.·Peng, B.

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Abstract

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. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/578080v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@75d1f3org.highwire.dtl.DTLVardef@12aafeforg.highwire.dtl.DTLVardef@184a1a5org.highwire.dtl.DTLVardef@90586a_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Evans, S., Lu, Z., McDonnell, L., Anderson, W., Peralta, F., Watkins, T., Ahmed, H., Luna-Flores, C. H., Loan, T. D., Navone, L., Trau, M., Scott, C., Speight, R., Vickers, C., Peng, B.. 2024-01-30. The integration of tandem gene repeats via a bacterial type-II toxin-antitoxin-mediated gene amplification (ToxAmp) system and stability visualisation in Saccharomyces cerevisiae. https://doi.org/10.1101/2024.01.30.578080

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