title: Generation of a Uracil Auxotroph Strain of the Probiotic Yeast Saccharomyces boulardii as a Host for the Recombinant Protein Production.
abstract: BACKGROUND
  Saccharomyces boulardii (S. boulardii) is the best known probiotic yeast. The genetic engineering of this probiotic strain requires the availability of appropriate mutants to accept various gene constructs carrying different selection markers. As the auxotrophy selection markers are under focus, we have generated a ura3 auxotroph mutant of S. boulardii for use in further genetic manipulations.
  
  
  METHODS
  Classical UV mutagenesis was used for the generation of auxotroph mutants. The mutants were selected in the presence of 5-FOA (5-Fluoroorotic acid), uracil and uridine. Uracil auxotrophy phenotype was confirmed by the ability of mutants to grow in the presence of uracil and the lack of growth in the absence of this compound. To test whether the uracil auxotrophy phenotype is due to the inactivation of URA3, the mutants were transformed with a plasmid carrying the gene. An in vitro assay was used for the analysis of acid and bile resistance capacity of these mutants.
  
  
  RESULTS
  Three mutants were found to be ura3 auxotroph as they were able to grow only in the presence of uracil. When the URA3 gene was added, these mutants were able to grow normally in the absence of uracil. Further in vitro analysis showed that the acid and bile resistance capacity of one of these mutants is intact and similar to the wild type.
  
  
  CONCLUSIONS
  A uracil auxotroph mutant of the probiotic yeast, S. boulardii, was generated and characterized. This auxotroph strain may have potential applications in the production and delivery of the recombinant pharmacuetics into the intestinal lumen.
authors.affiliation: Department of Food Hygiene, Faculty of Veterinary of Medicine, University of Tehran, Tehran, Iran.
authors.forename: Hassan
authors.initials: H
authors.lastname: Hamedi
authors.forename: Ali
authors.initials: A
authors.lastname: Misaghi
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Modarressi
authors.forename: Taghi Zahraei
authors.initials: TZ
authors.lastname: Salehi
authors.forename: Dorsa
authors.initials: D
authors.lastname: Khorasanizadeh
authors.forename: Vahid
authors.initials: V
authors.lastname: Khalaj
keywords: Probiotic; Recombinant proteins; Uracil; Yeasts