Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii

• Type of Material: Article
• Authors: Lauren E. Hudson, Emory University; Milo Fasken, Emory University; Courtney D. McDermott, Emory University; Shonna McBride, Emory University; Emily G. Kuiper, Emory University; David B. Guiliano, University of East London; Anita Corbett, Emory University; Tracey Lamb, Emory University
• Language: English
• Date: 2014-11-12
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: ©2014 Hudson et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0112660
• Title of Journal or Parent Work: PLoS ONE
• Volume: 9
• Issue: 11
• Start Page: e112660
• End Page: e112660
• Grant/Funding Information: Funding was provided through a CCVI Seed Grant from Emory University (http://www.emory.edu) and a New Innovator Award 1DP2AI112242-01 through the National Institutes of Health (http://commonfund.nih.gov/newinnovator/index).; The funders had no role in the study design, data collection and analysis, decision to publish, or preparation in the manuscript.
• Research Categories: Health Sciences, General; Health Sciences, Immunology

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.