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Development of a Dihydrofolate Reductase Selection System for Saccharomyces boulardii
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Development of a Dihydrofolate Reductase Selection System for Saccharomyces boulardii
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Journal Article
Development of a Dihydrofolate Reductase Selection System for Saccharomyces boulardii
2025
Overview
Saccharomyces boulardii, the only commercially available probiotic yeast, has gained attention as a recombinant live biotherapeutic product (rLBP) empowered with the expression of heterologous therapeutic proteins for treating gastrointestinal diseases. However, the genetic modification of S. boulardii intended for clinical use is hindered by regulatory and technical challenges. In this study, we developed a dihydrofolate reductase (DHFR)-based selection system as an innovative alternative to traditional auxotrophic selection strategies for engineering S. boulardii. The DHFR selection system overcame inherent resistance of the yeast to methotrexate (MTX) by incorporating sulfanilamide, a dihydrofolate synthesis inhibitor, to enhance selection efficiency. The system demonstrated robust functionality, enabling the efficient screening of high-expression clones and tunable expression of therapeutic proteins, such as cytokines and antibodies, by modulating MTX concentrations. Furthermore, the yeast’s endogenous DHFR homolog, DFR1, was shown to be a viable selection marker, providing greater host compatibility while maintaining functionality compared to DHFR. This selection system avoids reliance on foreign antibiotic selection markers and the construction of auxotrophic strains, thus simplifying engineering and allowing for a tunable protein expression. These advancements establish the DHFR/DFR1 selection system as a robust and versatile platform for developing S. boulardii-based live biotherapeutics.
Publisher
MDPI AG,MDPI
Subject
/ Cloning
/ Enzymes
/ Genes
/ Genetically modified organisms
/ Genomes
/ Proteins
/ Saccharomyces boulardii - drug effects
/ Saccharomyces boulardii - enzymology
/ Saccharomyces boulardii - genetics
/ Saccharomyces boulardii - metabolism
/ Tetrahydrofolate Dehydrogenase - genetics
/ Tetrahydrofolate Dehydrogenase - metabolism
/ Yeast
