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.

G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. GPCRs comprise the largest class of cell surface receptors in eukaryotes, and for more than three decades the pheromone-induced mating pathway in baker’s yeast Saccharomyces cerevisiae has served as a model for studying heterologous GPCRs (hGPCRs). Here we report transcriptome profiles following mating pathway activation in native and hGPCR-signaling yeast and use a model-guided approach to correlate gene expression to morphological changes. From this we demonstrate mating between haploid cells armed with hGPCRs and endogenous biosynthesis of their cognate ligands. Furthermore, we devise a ligand-free screening strategy for hGPCR compatibility with the yeast mating pathway and enable hGPCR-signaling in the probiotic yeast Saccharomyces boulardii. Combined, our findings enable new means to study mating, hGPCR-signaling, and cell-cell communication in a model eukaryote and yeast probiotics.

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12Saccharomyces boulardii is a widely used yeast probiotic which can counteract various 13 gastrointestinal disorders 1 . As a relative of Saccharomyces cerevisiae, S. boulardii exhibits rapid 14 growth and is easy to transform 2 and thus represents a promising chassis for the engineered 15 secretion of biomolecules. To establish S. boulardii as a platform for delivery of biomolecules to 16 the mammalian gut, we measured the amount and variance in protein expression enabled by 17 promoters, terminators, selective markers, and copy number control elements in this organism. 18These genetic elements were characterized in plasmidic and genomic contexts, revealing 19 strategies for tunable control of gene expression and CRISPR-mediated genome editing in this 20 strain. We then leveraged this set of genetic parts to combinatorially assemble pathways 21 enabling a wide range of drug and vitamin titers. Finally, we measured S. boulardii's residence 22 time in the gastrointestinal tracts of germ-free and antibiotic-treated mice, revealing the 23 relationships between dosing strategy and colonization level. This work establishes S. boulardii 24 as a genetically tractable commensal fungus and provides a set of strategies for engineering S. 25 boulardii to synthesize and deliver biomolecules during gut colonization. 26 27 abundant in the human gut, 21 and difficulty in producing high levels of post-translationally-42 modified proteins 22 . 43 44In addition to bacteria, a diverse fungal population also exists in the human gastrointestinal tract 45 (GIT) [23][24][25][26] . While the numerical abundance of fungal cells in the GIT is much lower than bacterial 46 cells, fungal cells are on average 100-fold larger, indicating that their biomass and role in the gut 47 may be larger than metagenomic surveys indicate 25,27 . Recent studies have shown that while 48 3 commensal fungi play important roles in the development of inflammatory bowel diseases, they 49 also provide protective heterologous immunity to pathogenic organisms by training the immune 50 system 28,29 . Furthermore, fungi are not susceptible to bacteriophage predation and are easily 51 engineered to secrete high titers of proteins that are post-translationally modified. Thus, in this 52 study, we established a pipeline to engineer the only eukaryotic probiotic that is approved by the 53 FDA: Saccharomyces boulardii 30 . 54 55 S. boulardii is a non-pathogenic yeast that was originally isolated from lychee and mangosteen in 56 1923 and has since been used to treat ulcerative colitis, diarrhea, and recurrent Clostridium 57 difficile infection 1,31 . S. boulardii is closely related to the famous budding yeast Saccharomyces 58 cerevisiae, indicating that it may be similarly amenable to engineering for the production of 59 biomolecules, including biologics requiring post-translational modifications 2,32 . Supporting this, 60 S. cerevisiae expression vectors can be transformed and propagated in S. boulardii, and 61 CRISPR/Cas9-mediated genome editing is functional in both ...

Directed evolution is a powerful method for engineering biology in the absence of detailed sequence-function relationships. To enable directed evolution of complex phenotypes encoded by multigene pathways, we require large library sizes for DNA sequences >5–10 kb in length, elimination of genomic hitchhiker mutations, and decoupling of diversification and screening steps. To meet these challenges, we developed Inducible Directed Evolution (IDE), which uses a temperate bacteriophage to package large plasmids and transfer them to naive cells after intracellular mutagenesis. To demonstrate IDE, we evolved a 5-gene pathway from Bacillus licheniformis that accelerates tagatose catabolism in Escherichia coli, resulting in clones with 65% shorter lag times during growth on tagatose after only two rounds of evolution. Next, we evolved a 15.4 kb, 10-gene pathway from Bifidobacterium breve UC2003 that aids E. coli’s utilization of melezitose. After three rounds of IDE, we isolated evolved pathways that both reduced lag time by more than 2-fold and enabled 150% higher final optical density. Taken together, this work enhances the capacity and utility of a whole pathway directed evolution approach in E. coli.