title: Stable expression plasmids for Streptomyces based on a toxin-antitoxin system.
abstract: BACKGROUND
  Bacteria included in the genus Streptomyces exhibit several attractive characteristics that make them adequate hosts for the heterologous expression of proteins. One of them is that some of its species have a high secretion capacity and hence the protein of interest could be released to the culture supernatant, facilitating downstream processing. To date, all the expression vectors described for these bacteria contain antibiotic resistance genes as selection markers. However, the use of antibiotics to produce proteins at industrial level is currently becoming more restricted owing to the possibility of contamination of the final product. In this report, we describe the use of the S. lividans yefM/yoeBsl toxin-antitoxin system to develop a stable plasmid expression system.
  
  
  RESULTS
  In order to use the yefM/yoeBsl system to stabilize expression plasmids in Streptomyces, a S. lividans mutant strain that contained only the toxin gene (yoeBsl) in its genome and the antitoxin gene (yefMsl) located in a temperature-sensitive plasmid was constructed and used as host. This strain was transformed with an expression plasmid harbouring both the antitoxin gene and the gene encoding the protein of interest. Thus, after elimination of the temperature-sensitive plasmid, only cells with the expression plasmid were able to survive. On using this system, two proteins - an α-amylase from S. griseus and a xylanase from S. halstedii - were overproduced without the addition of antibiotic to the culture medium. The production of both proteins was high, even after long incubations (8 days), and after serial subcultures, confirming the stability of the plasmids without antibiotic selection.
  
  
  CONCLUSIONS
  This is the first report that describes the use of a toxin-antitoxin system to maintain high -copy plasmids in Streptomyces. This finding could be a valuable tool for using Streptomyces as a host to produce proteins at the industrial and pharmaceutical levels without the use of antibiotics in the production step.
authors.affiliation: Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Salamanca 37007, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Sevillano
authors.forename: Margarita
authors.initials: M
authors.lastname: Díaz
authors.forename: Ramón I
authors.initials: RI
authors.lastname: Santamaría
mesh_terms: D000992:Antitoxins / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology; D048170:Streptomyces lividans / Q000378:metabolism*; D014118:Toxins, Biological / Q000235:genetics*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
chemical_list: D000992:Antitoxins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014118:Toxins, Biological; C484519:YefM protein, E coli; D000516:alpha-Amylases; D043364:Endo-1,4-beta Xylanases