Genetic Engineering of Auxotrophic Saccharomyces boulardii Mutants for Antigen Expression Open Access

Lee, Moon Young (2017)

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Probiotic yeast Saccharomyces boulardii has been identified as a well-suited potential vector for oral vaccine antigen delivery protection against infectious diseases; such a vector would ease the pain and cost of needlestick vaccines globally. In order to transform antigen DNA into S. boulardii without plasmids containing antibiotic resistance marker genes, functional auxotrophic S. boulardii mutants must be used. Currently, very few auxotrophic mutants of S. boulardii exist that grow as well as the wildtype and express functional recombinant proteins. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 gene editing system is a recently-developed technology that allows for selective mutation, making it favorable to make auxotrophic mutations in S. boulardii. The CRISPR-Cas9 system uses a specific CRISPR RNA to guide the Cas9 endonuclease to cut designated gene targets; this would only mutate the desired auxotrophic marker and no other gene. Here, we optimized the CRISPR-Cas9 gene editing system for yeast to generate leu2- auxotrophic mutants, ura3- auxotrophic mutants, and double auxotrophic mutants with both mutations (leu2- and ura3-) of S. boulardii. These mutants grow in low pH media and varying temperatures at comparable rates to the wildtype S. boulardii. The auxotrophic mutants were transformed with different proteins, including ovalbumin and interleukins. Importantly, using the double auxotrophic mutant, two recombinant proteins were successfully transformed and expressed, allowing for future application co-expressing an antigen and an adjuvant to enhance the function of the antigen. Overall, using the CRISPR-Cas9 system, we made effective auxotrophic single and double mutants S. boulardii that can be further tested for the development of an oral antigen delivery system.

Table of Contents

Table of Contents

Introduction 1-4

Methods 4-7

Results and Discussion 7-10

Conclusion 12

Acknowledgements 12

References 13-15

Tables and Figures 16-20

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