en 23626874 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. Conclusion: 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. Probiotic, Recombinant proteins, Uracil, Yeasts 29 34 https://www.ajmb.org/En/Article.aspx?id=106 https://www.ajmb.org/PDF/En/FullText/106.pdf HassanHamediDepartment of Food Hygiene, Faculty of Veterinary of Medicine, University of Tehran, Tehran, Iran451AliMisaghiDepartment of Food Hygiene, Faculty of Veterinary of Medicine, University of Tehran, Tehran, Iran452Mohammad HosseinModarressiDepartment of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran453TaghiZahraei SalehiDepartment of Microbiology, Faculty of Veterinary of Medicine, University of Tehran, Tehran, Iran454DorsaKhorasanizadehFungal Biotechnology Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran455VahidKhalajFungal Biotechnology Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran135