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Antibiotic-free selection plasmids in Streptomyces

AuthorsSevillano, Laura ; Díaz, Margarita ; Santamaría, Ramón I.
Issue Date9-Jul-2017
CitationFEMS 2017
Abstract[Backgrounds]: Streptomyces is a promising bacterial expression system that has been used to produce high levels of several proteins. In previous work, we described a new strategy for the stabilization of expression plasmids in Streptomyces based on the use of a toxin-antitoxin system (yefM/yoeBsl) as selection marker. We demonstrated the effectiveness of this system for the production of high levels of proteins without the use of antibiotics during the production step. However, the host used and the expression vectors still have the antibiotics resistance genes needed for its construction. [Objectives]: The objective of our work is the improvement of this system by the elimination of the antibiotic resistance genes still present in the host and in the expression plasmid. [Methods]: We have integrated the toxin gene (yoeBsl) in the genome with a new plasmid, pTES-tox, that has the target sites for the Cre recombinase (loxP) flanking the toxin gene and the phage attachment site (attP). Thus, after the expression of Cre recombinase we have deleted the plasmid backbone leaving only the toxin gene in the genome. In the same way, we have introduced the target sites for the Dre recombinase (rox) flanking the neomycin gene in the expression plasmid, the subsequent expression of the recombinase allowed us to delete it. [Conclusions]: We describe a system completely free of antibiotic resistance genes that is useful for the production of high yields of proteins in Streptomyces. This system could be a powerful tool for using Streptomyces as host to produce proteins at industrial and pharmaceutical level.
DescriptionResumen del trabajo presentado al 7th Congress of European Microbiologists, celebrado en Valencia (España) del 9 al 13 de julio de 2017.
Appears in Collections:(IBFG) Comunicaciones congresos
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