Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

Abstract

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

Metabolic Engineering of Probiotic Saccharomyces boulardii

“…During this assay, a pRS42K plasmid carrying the S. cerevisiae TDH3 promoter was used, indicating that the S. cerevisiae TDH3 promoter is working in S. boulardii. A previous study indicated that heterologous GFP can be expressed in S. boulardii (15). Here, mito-GFP and mito-RFP were functionally expressed and precisely localized to mitochondria, as expected, which means that even the localization of heterologous protein can be precisely achieved in S. boulardii.…”

“…Second, the efficiencies of genetic perturbations are as high as 100%, and target genetic perturbations are made due to the precise cutting and almost no off-target effects of the CRISPR-Cas9 system in yeast. This is a key advantage compared to UV mutagenesis (14,15), as the latter generates numerous unwanted mutations throughout the whole genome. Off-target mutation effects caused by CRISPR-Cas9 in mammalian systems have been reported previously (42,43).…”

“…However, genetic manipulation of S. boulardii has been limited due to the lack of auxotrophic mutants as well as concerns over the use of drug resistance genetic markers (13). Recently, mutations of URA3, the auxotrophic marker gene used most often in yeast genetics, have been generated in S. boulardii by UV mutagenesis (14,15) and the Cre-loxP system (16), paving the way for further engineering of this probiotic yeast. Nonetheless, there are several concerns over these approaches.…”

Probiotic Saccharomyces cerevisiae strains as biotherapeutic tools: is there room for improvement?

“…However, some transformation strategies require auxotrophic mutant strains, which lack the ability to synthesize an organic molecule, e.g., an amino acid, which can be restored upon transformation with a bicistronic plasmid encoding both a protein which will produce per turn that organic molecule and a desired foreign sequence to be expressed. So far, no natural auxotrophic mutants of S. boulardii have been reported and only recently auxotrophic strains of this probiotic yeast were obtained by UV mutagenesis (Hamedi et al 2013;Hudson et al 2014). Hamedi and colleagues elegantly described the first URA3 S. boulardii mutants (Hamedi et al 2013), obtained by mutagenesis of the wild-type strain lyo derived from DiarSafe, produced by the British company Wren Laboratories, Ltd.…”

Isolation and Characterization of Potential Probiotic Yeasts From Different Sources

“…Numerous studies have investigated the use of probiotic bacteria for the delivery of gastrointestinal therapeutics; however, eukaryotic probiotics have been less well studied. A major advantage of using probiotic yeast for this application is their ability as eukaryotes to create post-translational modifications that might enable expression of a wide variety of therapeutic proteins in their proper conformation [10]. Live Saccharomyces cerevisiae cells are employed as biotherapeutic or probiotic agents for re-equilibration of intestinal microflora, and the latest studies have demonstrated their efficacy in treating chronic or recurrent diarrhea, especially in those cases associated with clostridium difficile [11].…”