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Title

Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces

AuthorsSevillano, Laura ; Díaz, Margarita ; Santamaría, Ramón I.
KeywordsHeterologous protein expression
Antibiotic marker-free
Streptomyces
Toxin-antitoxin
Separate-component-stabilization system
Issue Date2017
PublisherBioMed Central
CitationMicrobial Cell Factories 16: 164 (2017)
Abstract[Background]: The industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential.
[Results]: In this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed.
[Conclusions]: The total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described.
DescriptionThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License.
Publisher version (URL)https://doi.org/10.1186/s12934-017-0781-y
URIhttp://hdl.handle.net/10261/156340
DOI10.1186/s12934-017-0781-y
E-ISSN1475-2859
Appears in Collections:(IBFG) Artículos
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