In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii

Abstract

Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (β-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1−2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled β-carotene synthesis (194 μg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional β-carotene recovered in feces was 56-fold higher than the β-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.

The Role of β-Carotene in Colonic Inflammation and Intestinal Barrier Integrity

“…β-Carotene concentration can be between 680 and 2,255 nM in circulation depending on whether it is consumed as a pure compound or within the foods (54,56). We recently reported that engineered S. boulardii synthesized high doses of β-carotene in the colon of mice, and the intake of such engineered probiotics may result in an even higher β-carotene concentration in the gut (57). Altogether, the dosage of β-carotene used in this study is within physiological relevance and can be achieved by humans.…”

Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts

“…S2A-D) compared to S. cerevisiae. Indeed, from understanding the regulatory mechanisms of the mating pathway, and taking advantage of S. boulardii's growth, survival, and residence time in the gastrointestinal tract as compared to S. cerevisiae 36 , we envision that probiotic S. boulardii can now be engineered to dynamically respond to disease markers for timely and accurate secretion of therapeutic compounds, and as such adds an important capability to its already established therapeutic potential. Extending from this we furthermore foresee this study to foster the development of growth-based high-throughput screens of hGPCR-mediated chemotropism within environmental engineering, MAPK pathway signaling, drug discovery for therapeutic purposes, and enzyme optimization for metabolic engineering and biotechnology.…”

“…pDAM123 was made by assembling fragments from gDAM15 (α-leader secretion signal and P-factor) ampli ed with DAM76+DAM78, P TEF1 ampli ed with 1564+1565, and T CYC1 ampli ed with DAM80+DAM81, into pCfB2899. Plasmid pJV452 was constructed by Gibson (NEB) assembly of pEDJ400 linearized with oligos JV498+JV499, which excluded the 2μ element, and CEN/ARS from plasmid DD118 36 ampli ed with oligos JV500+JV501. Plasmid pDAM194 containing P FUS1 -yEGFP-T FUS1 was made by ampli cation of gDNA from strain CPK16 with oligos JV502+JV503, which was then assembled into plasmid pJV452.…”

“…P-factor producing strains were created by integration of NotI-digested pDAM123 using gRNA plasmid pCfB3020, which was done in strain CPK152 and SBY55, resulting in CPK448 and SBY155, respectively. Strain engineering of S. boulardii: Construction of ura3/his3 auxotrophies: Transformation, Incubations, and recovery after transformation were done at 37 o C as previously described 36 . S. boulardii ATCC MYA-796 (Sb.MYA-796) was co-transformed with 1 µg of gRNA_URA3+Cas9 plasmid pDD111 and 1.5 µg of repair template DDgb009 ampli ed with oligos DDpr273 and DDpr274.…”

The development of live biotherapeutics against Clostridioides difficile infection towards reconstituting gut microbiota

“…S. boulardii may also be able to deliver other therapeutics, including bacteriocin, cytokines, peptides, and small molecular drugs. 120–123 In order to improve the yield of target cargoes, the components of the gene cassettes, such as signal peptides, promotors/terminators, selection markers, etc., and maintenance of the exogenous gene of interests as plasmids or chromosomal integration have been modified in those studies. Among them, Chen et al and Liu et al have demonstrated the in vivo activity of the yeast-secreted heterologous proteins after passing through animal GI tract.…”