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 develop...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.展开更多
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 facilita...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-leakyβ-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.展开更多
基金supported partially by the Australian Government through the Australian Research Council Centres of Excellence funding scheme(project CE200100029)。
文摘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.
基金supported by the Australian Government through the Australian Research Council Centres of Excellence funding scheme(project CE200100029).
文摘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-leakyβ-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.
