Methods of treating gastrointestinal disorders with engineered yeast

Abstract

Provided herein are engineered Saccharomyces boulardii (S. boulardii) yeasts. The engineered yeasts comprise an exogenous protein encoded by a heterologous agglutinin polynucleotide linked to a target domain polynucleotide encoding a target domain. The target domain is encoded in frame to allow for production of the exogenous protein, and the exogenous protein is displayed on the surface of, or is secreted from, the yeast. Also provided are compositions comprising the engineered yeast, methods of treating a gastrointestinal or proliferative disorder, methods of detecting a gastrointestinal or proliferative disorder within the gastrointestinal tract, and kits for preparing an engineered S. boulardii yeast.

Description

[0001] METHODS OF TREATING GASTROINTESTINAL DISORDERS WITH ENGINEERED YEAST

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 730,664, filed on December 11, 2024, the contents of which are incorporated by reference herein in their entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under P30 GM145393 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] A Sequence Listing accompanies this application and is submitted as an XML file of the sequence listing named “16985200193_SL. xml” which is 93,628 bytes in size and was created on December 11, 2025. The sequence listing is electronically submitted via Patent Center and is incorporated herein by reference in its entirety.

[0008] BACKGROUND

[0009] Gastrointestinal diseases caused by bacteria are a global issue, many of which lack adequate therapeutic treatment options. One solution may be the use of probiotic organisms that can express therapeutic molecules. Surface display systems, which enable immobilization of therapeutic molecules on the surface of microorganisms, have been developed using S. cerevisiae, a yeast commonly used in biotechnology applications and food production. However, the use of S. cerevisiae for oral administration is limited by its sensitivity to gastric acid, bile salts, and temperature. Thus, there remains a need for improved methods of treating gastrointestinal disorders, along with improved microorganisms that are capable of displaying therapeutic molecules and that can be used to treat and / or detect gastrointestinal conditions.

[0010] SUMMARY

[0011] In a first aspect, the disclosure provides an engineered Saccharomyces boulardii (S. boulardii)' yeast comprising an exogenous protein. The exogenous protein (1) is encoded by a heterologous agglutinin polynucleotide linked to a target domain polynucleotide encoding a target domain in frame to allow for production of the exogenous protein, and (2) is displayed on the surface of or released from the yeast. In some embodiments, the exogenous protein comprises a target domain linked to an Aga2 domain, with the Aga2 domain conjugated to an Agal protein via at least one disulfide bond. The target domain may be an antibody or a single-chain variable fragment and may be linked by a linker peptide to the Aga2 domain. The exogenous protein may further comprise a detectable tag, including an affinity tag, an epitope tag, and / or a fluorescent peptide or marker, and may be configured to bind to a target associated with a gastrointestinal disorder. The engineered yeast may be an auxotrophic yeast, including a URA3 auxotroph. The engineered yeast may be stable in gastrointestinal conditions. The heterologous polypeptide may be integrated into the S. boulardii genome and may be comprised within an a-agglutinin surface display construct derived from Saccharomyces cerevisiae.

[0012] In a second aspect, provided herein are compositions comprising the engineered yeast and a pharmaceutically acceptable carrier. The composition may comprise 1 million to 20 billion colony forming units of the engineered yeast per dose and may be formulated for oral administration.

[0013] In a third aspect, provided herein are methods of treating a subject having or suspected of having a gastrointestinal or proliferative disorder. The methods comprise administering to the subject the engineered yeast or the composition comprising the engineered yeast. The composition may be administered orally, and the gastrointestinal disorder may be associated with a pathogen and / or a pathogenic toxin. In some embodiments, the target domain binds to the pathogen or the pathogenic toxin, and the binding may neutralize and or facilitate gastrointestinal clearance of the pathogen or pathogenic toxin. In some embodiments, the disorder is a proliferative disorder, and the target domain is a therapeutic agent to treat the proliferative disorder. The proliferative disorder may be a cancer, including colorectal cancer. The target domain may comprise a therapeutic agent and a mucus binding agent, which mucus binding agent may maintain the yeast in the lower gastrointestinal tract longer than a reference yeast without the mucus binding agent.

[0014] In a fourth aspect, provided herein are methods of detecting a gastrointestinal disorder or proliferative disorder within the gastrointestinal tract of a subject. The methods comprise (a) orally administering to the subject the engineered yeast or the composition comprising the engineered yeast, and (b) detecting the engineered yeast, wherein the detection, location or duration of the yeast in the gastrointestinal tract is indicative of a disorder. The gastrointestinal disorder or proliferative disorder may be a cancer, including colorectal cancer. The detecting may be performed by in vivo fluorescent imaging or magnetic resonance imaging.

[0015] In a fifth aspect, provided herein are kits for preparing an engineered 5. bonlardii yeast comprising an auxotrophic S. bonlardii yeast and an exogenous a-agglutinin surface display expression construct derived from Saccharomyces cerevisiae (S. cerevisiae). The expression construct comprises (i) a cloning site to allow for cloning of a target domain linked to an Aga2 domain and an Agal domain and (ii) promoters to allow expression of both the Agal domain and the target domain linked to the Aga2 domain. The expression construct may be capable of being integrated into a chromosome of the yeast, and the target domain may comprise a detectable tag, including an affinity tag, an epitope tag, and / or a fluorescent peptide or marker. The target domain may be configured to bind to a target associated with a gastrointestinal disorder or proliferative disorder. The auxotrophic yeast may be a URA3 auxotroph, and the engineered yeast may be stable in gastrointestinal conditions.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] FIGS. 1A-1D illustrate S. bonlardii strain generation. (1 A) PCR verification of URA3 gene knockout ( / / / 773A). This strain is derived from a commercial probiotic strain of S. bonlardii (strain DBVPG 6763). The table gives a key indicating theoretical band sizes for each gene cassette amplified with the corresponding primer pair. A different antibiotic resistance cassette was used to knock out each URA3 allele. Both contain the Tefl promoter and are of different sizes, indicated by two PCR products from the “TF” primer set in the figure. The second gel panel shows the absence of large molecular weight bands corresponding to antibiotic cassette elimination. (IB) DNA gel showing removal of the plasmid containing the gene for Cre recombinase. (1C) Graphic (Snapgene) illustrating the antibody surface display components using the anti-FAP example. AGA J and AGA2 are the a-agglutinin genes, TEF1 and GAP (TDH3) are the promoters, HA is the haemagluttinin tag, and scFv is the anti-FAP single-chain fragment variable antibody. (ID) PCR verification of the antibody display cassette integration using primer pairs flanking the insertion region (XI-3 or XII-5) Control is the IIRA3A strain from FIG. 1 A, and anti-FAP and anti-Lys are strains with integrated expression cassettes at the indicated chromosomal targets.

[0019] FIG. 2 illustrates strain validation. Each strain was sequenced with Oxford Nanopore Technology long-read sequencing and aligned to the S. cerevisiae S288C genome. Three genes were evaluated for mutations reported as genotypes unique to 5. boulardii. Integrated Genomics Viewer was used to visualize the comparison with the reference S. cerevisiae genome. The nucleotide sequence at the bottom of each box represents the reference sequence. The horizontal grey bars represent a subset of sequencing reads for each sample. Red highlight (denoted with *) indicates a mutation in the sequenced strand to thymine, orange (denoted withA) to guanine, green (denoted with +) to adenine, and blue (denoted with ~) to cytosine. The forward strand is shown in each case, meaning that the sense strand is represented for WHI2 and the antisense strand is represented for PGM2 and SDH1. See also FIG. 6 for sequence alignments of WHI2, PGM 2. and SDH1.

[0020] FIGS. 3A-3E illustrate an antibody functionally displayed on the surface of S. boulardii. (3 A) Illustration (Biorender) of the yeast surface display system used in this study. The HA tag on the Aga2-antibody fusion protein is recognized by an anti -HA antibody labelled with a fluorophore and the antigen (circle) is labelled with a different fluorophore. (3B) Yeast cells displaying antibody by the Agal / Aga2 system were treated with reducing agents, then the HA tag was probed and detected by flow cytometry to demonstrate the disulfide-dependent display of the fusion protein. (3C) A Western blot was performed on the parental strain and two strains containing genomic insertion of the surface display cassette to detect fusion protein expression (see FIG. 8) for additional details). (3D) Yeast cells with (anti-FAP, anti-Lys) or without (Control) the antibody display cassette were evaluated by flow cytometry. (3E) Confocal microscopy was used to visualize FAP and anti -HA binding to the anti-FAP strain. The white scale bar represents 10 pm in length.

[0021] FIGS. 4A-4B illustrate functional antibody surface expression in simulated colonic fluids. Yeast cultures were grown overnight at 37°C in the indicated media, then stained with fluorescent conjugates of both an anti -HA antibody and antigen. The Control and anti -Lysozyme strains were presented with lysozyme and the anti-FAP strain was presented with FAP. Each sample was additionally stained with Sytox blue viability stain. This experiment was performed twice with replicates. (4A) The proportion of cells staining negative for Sytox blue in each sample were graphed to determine the fraction that was viable after overnight incubation in various fluids. The flow cytometry histogram to the right shows an example of the viability stain detection strategy, where gate R5 was used to derive “viable” cells measured in the bar graph. Data are represented as mean + / - SD of 3 replicates. (4B) Bivariate plots are indicative of cell surface antibody expression (HA tag) and / or antigen binding (Antigen). Percentages indicate the population percentage allocated to each quadrant. Row labels indicate the overnight incubation medium and column labels indicate the yeast strain and medium in which the cells were incubated with antibody and antigen. Fed or FeSSCoF indicates fed-state simulated colonic fluids and Fasted or FaSSCoF indicates fasted-state simulated colonic fluids.

[0022] FIGS. 5A-5C illustrates surface binding display measurements. (5A) Yeast cells were stained with serial dilutions of anti-HA antibody (top) or fluorescently labelled FAP (bottom) for 1 hour and analyzed by flow cytometry. MFI was plotted versus concentration using a 4 parameter least squares fit in GraphPad to estimate ECso values. (5B) The number of antibody molecules expressed per cell was estimated using flow cytometry. A standard curve was created with BD Quantibrite beads conjugated to known amounts of PE molecules and used to analyze anti-HA PE antibody conjugates bound to yeast cells. Data is shown as a histogram measuring intensity in the PE channel comparing beads and yeast expressing a fusion protein or not (Control). (5C) The line graph shows antibody per cell quantification of each strain at each time point based on the bead- determined standard curve and background correction. Data are represented as mean + / - SD of 3 replicates. MFI = median fluorescence intensity, FAP = fibroblast activation protein, HA = hemagglutinin affinity tag, PE = phycoerythrin, Sat. = saturation.

[0023] FIG. 6 illustrates the alignment of three genes from S. boulardii, PGM2 (SEQ ID NO: 30), SDH1 (SEQ ID NO: 28), and WHI2 (SEQ ID NO: 26), to the 5. cerevisiae strain S288C reference PGM2 (SEQ ID NO: 29), SDH1 (SEQ ID NO: 27), and WHI2 (SEQ ID NO: 25) sequences, related to FIG. 2. Snapgene software was used to align the consensus sequence across the WGS reads for each S. boulardii strain (consensus) to the sequence found in S. cerevisiae strain S288C (reference). Dots in the “consensus” line represent identical bases relative to the “reference.” Differences representing mutations are specified.

[0024] FIG. 7 illustrates sequencing confirmation of genetic changes, related to FIG. 2. Whole genome sequencing via long-read sequencing technology was used to confirm knockout of the URA3 gene as well as insertion of the antibody expression cassette in two different strains, described in FIG. 1. This figure shows each of three strains aligned in the genome browser IGV and evaluated in the context of the surrounding annotated reference genome from S. cerevisiae. This view is detailed in FIG. 2.

[0025] FIG. 8 shows uncropped western blot data, related to FIG. 3C. Cell lysates were separated by electrophoresis, transferred to a PVDF membrane, and probed first with anti-HA tag antibody conjugated to iFluor 650 and imaged. The blot was next probed with anti-Gapdh antibody, then with anti-mouse antibody conjugated to Alexa Fluor 647. The band attributed to the fusion protein containing the HA tag runs slightly above the 50 kDa marker, with a predicted molecular weight of about 38 kDa suggesting potential post-translational modification. Black arrows indicate the bands presented in FIG. 3. Only the whole cell lysis lanes are shown in FIG. 3.

[0026] DETAILED DESCRIPTION

[0027] Despite posing a worldwide health concern, many gastrointestinal diseases and disorders caused by bacteria currently lack adequate therapeutic treatments or non-invasive diagnostic tests. One solution to this may be the use of probiotic organisms that can express therapeutic molecules or diagnostic molecules. Surface display systems are genetic engineering tools that allow the expression of heterologous proteins on the cell surface of microorganisms such as yeast8. These systems enable the immobilization of enzymes, antibodies, and other biomolecules on the cell surface, which can facilitate their characterization and evolution9Several surface display systems have been developed for S. cerevisiae, one of the most used species of yeast for biotechnology applications as well as food production. However, 5. cerevisiae is not an ideal candidate for oral administration, as it is sensitive to gastric acid and bile salts and does not tolerate body temperature very well.

[0028] Saccharomyces boulardii (S. boulardii) is a non-pathogenic yeast that has been widely used as a probiotic for the prevention and treatment of various gastrointestinal disorders such as antibiotic-associated diarrhea, inflammatory bowel disease, and Clostridium difficile infection1,2. The beneficial effects of S. boulardii are attributed to its ability to modulate the intestinal microbiota, enhance the mucosal barrier function, and modulate the immune response. In addition to being a tested probiotic, it is a species of budding yeast closely related to Saccharomyces cerevisiae. Because of this, there are a host of molecular tools and methods available for modifying this species, making it a prime candidate for exploring the possibility of generating a genetically modified probiotic. This includes the potential of S. boulardii as a platform for delivering therapeutic biomolecules to the gut, which remains largely unexplored, but may be useful particularly when it comes to secreting antibodies or other molecules into the gut3 7.

[0029] In contrast to S. cerevisiae, S. boulardii is more resistant to harsh gastrointestinal conditions and has a proven safety record as a probiotic. Therefore, utilizing a surface display system in S. boulardii may offer a way to safely and effectively generate a therapeutic outcome or provide diagnostic information in the gut. In contrast to related options such as secretion of neutralizing antibodies by S. boulardii, surface display possesses the potential advantage of a better effective affinity and removal of toxin proteins due to the avidity effect of antibodies grouped on the cell surface and the fact that this system would inherently move these captured proteins away from the site of infection as the yeast cell passes through the gastrointestinal tract (GIT).

[0030] In the Examples, the inventors demonstrate the application of a functional surface display system in an engineered S. boulardii yeast. The surface display system, which is based on the a- agglutinin cell-surface anchoring system from S. cerevisiae, efficiently displays a genetically encoded single-chain antibody and retains its functionality in simulated intestinal fluids. Thus, provided herein are engineered S. boulardii yeasts, compositions comprising the engineered yeasts, and kits for preparing an engineered 8. boulardii yeast. Also provided are methods of using the engineered 8. boulardii, including methods of treating a gastrointestinal or proliferative disorder, and methods of detecting a gastrointestinal or proliferative disorder.

[0031] Engineered Yeasts

[0032] The present disclosure provides engineered S. boulardii yeasts. In an aspect, the engineered yeast comprises an exogenous protein. The engineered yeast include a heterologous agglutinin polynucleotide linked to a target domain polynucleotide in frame to allow expression of the exogenous protein on the surface or released from the engineered yeast. As used herein, the terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand). The polynucleotides may be synthetic DNA, cDNA or genomic DNA. Polynucleotides homologous to the polynucleotides described herein are also provided. Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide.

[0033] As used herein, “a heterologous agglutinin polynucleotide” refers to a polynucleotide encoding an agglutinin, which is a cell adhesion glycoprotein capable of causing particles to aggregate and / or coagulate. Any suitable heterologous agglutinin polynucleotide may be used, including without limitation, a-agglutinin or a-agglutinin polynucleotide. The heterologous agglutinin polynucleotide is linked to a target domain polynucleotide encoding a target domain. In some embodiments, the heterologous agglutinin polynucleotide is linked to the target domain polynucleotide by a linker sequence encoding a linker peptide. The target domain is encoded in frame with the agglutinin polypeptide, allowing for production of the exogenous protein by the yeast. After production, the exogenous protein is displayed on the surface of the yeast or is secreted or released from the yeast.

[0034] In some embodiments, the heterologous agglutinin polynucleotide protein is provided as part of an expression construct in which it is operably linked to a promoter. As used herein, the term “expression construct” refers a to recombinant polynucleotide, e.g.., a polynucleotide that was formed by combining at least two polynucleotide components from different sources, natural or synthetic. For example, an expression construct may comprise the coding region of one gene operably linked to a promoter that is (1) associated with another gene found within the same genome, (2) from the genome of a different organism, or (3) synthetic. Expression constructs can be generated using conventional DNA recombination methods. The expression construct may allow for various levels of expression of the exogenous protein. In some embodiments, the engineered yeast comprises at least 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 copies of the exogenous protein, or a number of copies of the exogenous protein within a range bounded by any of the foregoing. In the Examples, the inventors demonstrate the system may be used to provide over 3000 copies of an exogenous protein / cell. The copy number will vary depending on the exogenous protein being expressed, the promoter and type of expression construct used as will be understood to those of skill in the art.

[0035] As used herein, the term “promoter” refers to a DNA sequence that defines where transcription of a polynucleotide begins. RNA polymerase and the necessary transcription factors bind to the promoter to initiate transcription. Promoters are typically located directly upstream (i.e., at the 5' end) of the transcription start site. However, a promoter may also be located at the 3’ end, within a coding region, or within an intron of a gene that it regulates. Promoters may be derived in their entirety from a native or heterologous gene, may be composed of elements derived from multiple regulatory sequences found in nature, or may comprise synthetic DNA. A promoter is “operably linked” to a polynucleotide if the promoter is positioned such that it can affect transcription of the polynucleotide. Suitable promoters for use with the present invention include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters. In some embodiments, the heterologous agglutinin polynucleotide comprises a bidirectional TDH3(GAP) / TEF1 promoter from S. cerevisiae. Those of skill in the art understand how to select an appropriate promoter to drive expression of the exogenous proteins described herein.

[0036] The heterologous agglutinin polynucleotide and / or expression construct may be introduced into the engineered yeast using any suitable method known in the art, including, without limitation, bacteriophage or viral infection, electroporation, heat shock, lipofection, microinjection, and particle bombardment. In some embodiments, the heterologous agglutinin polynucleotide and / or expression construct is comprised within a vector. As used herein, a “vector” is any particle used as a vehicle to artificially carry an exogenous nucleic sequence, typically DNA into another cell, where it can be replicated and / or expressed. Suitable vectors are known in the art, including, without limitation, plasmids, viral vectors, cosmids, and artificial chromosomes. In some embodiments, the heterologous agglutinin polynucleotide and / or expression construct is integrated into the S. boulardii genome. The heterologous agglutinin polynucleotide can be inserted randomly into the genome or targeted to a specific location (e.g., via homologous recombination). In some embodiments, the heterologous agglutinin polynucleotide and / or expression construct is integrated into the S. boulardii genome at a genomic location. The location may be within chromosome XI and / or XII.

[0037] In the Examples, the inventors utilized an a-agglutinin-based surface display system derived from S. cerevisiae. a-Agglutinin is a heterodimer composed of two subunits: an Aga2 protein, which exhibits binding activity, and an Agal protein, which anchors the Agal protein to the cell surface via two disulfide bonds. Thus, in some embodiments, the heterologous agglutinin polynucleotide is comprised within an a-agglutinin surface display construct derived from S. cerevisiae. In some embodiments, the exogenous protein comprises the target domain linked to an Aga2 domain, and the Aga2 domain is conjugated to an Agal protein via at least one disulfide bond. Tn other embodiments, the target domain is directly linked to the Agal protein. In some embodiments, the target domain is linked to the Aga2 domain or the Agal protein by a linker peptide. The linker peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues. The linker peptide may comprise any amino acid sequence that does not substantially hinder the interaction of the target domain with its corresponding target molecule(s). In some embodiments, the linker is a (GGGGS)3 linker (SEQ ID NO: 31). The exogenous protein may comprise a protease cleavage site (e.g., a Factor Xa site) between the target domain and the Aga2 domain. In other embodiments, the exogenous protein does not comprise a protease cleavage site between the target domain and the Aga2 domain. In some embodiments, the engineered yeast comprises one or more nucleic acids comprising a sequence as set forth in any one of SEQ ID NOs: 1-5, or having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 1-5. In some embodiments, the Agal protein comprises the amino acid sequence set forth in SEQ ID NO: 6, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6. In some embodiments, the Aga2 domain comprises the amino acid sequence set forth in SEQ ID NO: 7, or comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7.

[0038] As used herein, a “target domain” refers to a protein or peptide that is capable of recognizing and / or interacting with a target molecule or a domain capable of eliciting a therapeutic effect or having a use as a diagnostic for a disease or condition. A skilled artisan will readily appreciate proteins and peptides which may suitably be used to recognize and / or interact with a target molecule. In some embodiments, the target domain is an enzyme. In some embodiments, the target domain is an antibody. An “antibody” refers to a protein that comprises at least one antigen-binding domain from an immunoglobulin molecule. The antibody may be, without limitation, a whole antibody (e.g., IgG, IgA, IgE, IgM, IgD), a chimeric antibody, or an antibody fragment, including a single chain variable fragment (scFv). In some embodiments, the target domain is a scFv. As further described in the methods section, the engineered yeast may be utilized in methods of detecting and / or treating a gastrointestinal disorder or proliferative disorder. Thus, in some embodiments, the exogenous protein and / or the target domain is configured to bind to a target associated with a gastrointestinal disorder. A skilled artisan will be able to select a target domain based on its ability to selectively recognize and / or interact with a target molecule associated with the gastrointestinal or proliferative disorder to be detected / treated. In some embodiments, the target domain binds to the target molecule with a half maximal effective concentration (EC50) of less than 500 nM, 250 nM, 100 nM, 50 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.25 nM, or 0.1 nM or with a EC50 within a range bounded by any of the foregoing.

[0039] In some embodiments, the exogenous protein further comprises a detectable tag. As used herein, a “detectable tag” is a peptide or marker that is genetically grafted onto the exogenous protein, and which facilitates detection of the exogenous protein. Suitable detectable tags include, without limitation, affinity tags (e.g., chitin binding protein (CBP), maltose binding protein (MBP), Strep, glutathione-S-transferase (GST), poly(His) tags), solubilization tags (e.g., thioredoxin (TRX), poly(NANP), MBP, GST), epitope tags for antibody-based detection (e.g., ALFA-tag, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag, NE-tag), and fluorescent tags (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP)), and enzymatic tags (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose-6-phosphatase, acetylcholinesterase) for visual detection. The detectable tag may be detectable by any suitable method, including, without limitation, enzyme-linked immunoassay (ELISA), dot blotting, western blotting, flow cytometry, mass spectrometry, fluorescence microscopy, magnetic resonance imaging, and chromatographic methods.

[0040] Any suitable strain of S. boulardii may be used to generate the engineered yeast. In some embodiments, the engineered yeast is a commercially available S. boulardii strain DBVPG 6763. In some embodiments, the engineered yeast is auxotrophic. As used herein, “auxotrophic” refers to an organism that is unable to synthesize one or more compounds that are required for growth of the organism. Utilizing an auxotrophic probiotic strain may be desirable in developing a genetically engineered therapeutic, to prevent rampant proliferation in the environment. A skilled artisan will readily identify mutations known in the art to yield auxotrophic S. boulardii strains. In some embodiments, the engineered yeast is a URA3 auxotroph.

[0041] As further described in the methods section, the engineered yeast may be utilized in methods of detecting and / or treating a gastrointestinal disorder or proliferative disorder. Thus, in some embodiments, the engineered yeast is stable in gastrointestinal conditions. The stability of the engineered yeast may be assessed using any suitable method known in the art. In the Examples, samples of the engineered yeast were cultured overnight at 37°C in either water, yeast media (YPD), Fasted-State Simulated Colonic Fluids (FaSSCoF) or Fed-State Simulated Colonic Fluids (FeSSCoF). Following incubation, samples were stained with propidium iodide and analyzed via flow cytometry. In some embodiments, the engineered yeast exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, or 99% viability following exposure to gastrointestinal conditions for at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 2 days, 7 days, or 14 days.

[0042] In some embodiments, the exogenous protein further comprises a mucus binding agent and / or a collagen binding agent. As used herein, a “mucus binding agent” refers to a protein or peptide capable of interacting with mucus in the gastrointestinal tract, and a “collagen binding agent” refers to a protein or peptide capable of interacting with collagen in the gastrointestinal tract. Mucus binding agents and / or collagen binding agents can be found on the cell surface of some bacteria, including Lactobacillus reuteri and Lactobacillus plantarum. In some embodiments, the mucus binding agent is mucin-binding protein (MUB). In some embodiments, the mucus binding agent comprises SEQ ID NO: 9, or a peptide having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the collagen binding agent is collagen-binding protein Cnb. In some embodiments, the collagen binding agent comprises SEQ ID NO: 8, or a peptide having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the mucus binding protein maintains the engineered yeast in the lower gastrointestinal tract longer than a reference yeast which does not include the mucus binding agent. In some embodiments, the engineered yeast is maintained in the lower gastrointestinal tract for at least 6 hours, 12 hours, 24 hours, 2 days, 7 days, or 14 days, or for a period of time within a range bounded by any of the foregoing.

[0043] Compositions

[0044] The present disclosure also provides compositions comprising the engineered yeast described herein and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” is any solid or liquid filler, diluent or encapsulating material suitable for in vivo administration. Suitable pharmaceutically acceptable carriers are known in the art and include, without limitation, sugars, such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other nontoxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions, according to the desires of the formulator. In some embodiments, the composition is formulated to include an acid-resistant coating. Suitable acid- resistant coatings are known in the art and include, without limitation, methacrylate, cellulose polymers, and polyvinyl acetate phthalate.

[0045] In some embodiments, the composition may also comprise other suitable agents such as a stabilizing delivery vehicle, carrier, support or complex-forming species. The coordinate administration methods and combinatorial formulations of the instant invention may optionally incorporate effective carriers, processing agents, or delivery vehicles, to provide improved formulations for delivery of the engineered yeasts described herein. In some embodiments, the composition comprises a freeze-dried powder.

[0046] The composition may comprise any suitable amount of the engineered yeast. In some embodiments, the composition comprises an "effective amount" or “therapeutically effective amount” of the engineered yeast, which means an amount sufficient to effect beneficial or desirable biological and / or clinical results. In some embodiments the composition comprises about 0.25 million, 0.5 million, 1 million, 10 million, 50 million, 100 million, 500 million, 1 billion, 5 billion, 10 billion, 15 billion, 20 billion, 25 billion, or 50 billion colony forming units of the engineered yeast, or an amount of the engineered yeast within a range bounded by any of the foregoing. In some embodiments, the composition comprises 1 million to 20 billion CFUs of the engineered yeast. The composition may comprise one dosage form or more than one dosage form.

[0047] The composition may be formulated for any suitable route of administration. In some embodiments, the composition is formulated for oral administration. The composition may be in liquid form, powder form or in a pressed tablet form.

[0048] Methods of Treatment The present disclosure also provides methods of treatment, comprising administering the engineered yeasts or compositions described herein to a subject having or suspected of having a gastrointestinal or proliferative disorder. As used herein, the terms “treating,” “treatment,” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. In some embodiments, the engineered yeast or composition comprising the engineered yeast is used in combination with one or more additional therapeutic agents.

[0049] The “subject” or “patient” can be any organism in need of and / or subjected to a treatment, such as a farm animal, domestic animal, or human. In some embodiments, the subject is a human. The engineered yeast or composition may be administered by any suitable route, including, but not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, and parenteral administration, including injectable such as intramuscular administration, intertumoral administration, intradermal administration, and subcutaneous administration. In some embodiments, the engineered yeast or composition is administered orally. In some embodiments, the exogenous protein is delivered to the lower gastrointestinal tract of the subject following administration. As used herein, the “lower gastrointestinal tract” refers to the final part of the digestive system, beginning at the cecum and extending to the anus.

[0050] In some embodiments, the gastrointestinal disorder is associated with a pathogen and / or a pathogenic toxin. As used herein, a “pathogen” is any microorganism capable of causing disease in a patient or subject. Any suitable pathogen may be treated using the disclosed methods, including, for example, a bacterium, a fungus, a virus, or a protist. Exemplary pathogens to be treated using the methods include, without limitation, Bacillus, Escherichia, Campylobacter, Citrobacter, Clostridium, Clostridium difficile, Enterotoxigenic E.coli (ETEC), Enteroaggregative Escherichia coli (EAggEC), Shiga-like Toxin producing E.coli, Helicobacter, Klebsiella, Listeria, Staphylococcus, Salmonella, Shigella, Vibrio, Yersinia, Yersinia enterocolitica, Adenovirus, Norovirus, Rotavirus A, Cryptosporidium parvum, Entamoeba histolytica, Giardia lamblia, Clostridia, Staphylococcus aureus, Klebsiella pneumonia, influenza, Zika, dengue, chikungunya, West Nile virus, Japanese encephalitis, malaria, HIV, H1N1, HPV, Hepatitis, Ebola, Streptococcus, Neisseria, and Mycobacterium. As used herein, a “pathogenic toxin” refers to a substance produced and / or secreted by a pathogen that causes disease in an infected subject by targeting and / or interacting with the subject’s cells. Any suitable pathogenic toxin may be treated using the disclosed methods, including, without limitation, Shiga toxin, E. coli heat-labile toxin, E. coll heat-stable toxin, Salmonella enterotoxin, C. difficile toxin A, C. difficile toxin B, a cholera toxin, a staphylococcal enterotoxin, cereulide, a & cereus enterotoxin, listeriolysin O, a cytolethal distending toxin, and vacuolating cytotoxin A.

[0051] In some embodiments, the target domain binds to the pathogen or the pathogenic toxin. The target domain may be capable of selectively binding the pathogen or the pathogenic toxin. A target domain is said to “selectively” bind when it is capable of binding to a pathogen (e.g., to the surface of a pathogenic cell) or pathogenic toxin, but has substantially reduced binding to other cell types or molecules. In some embodiments, the target domain binds to the pathogen or the pathogenic toxin with a half maximal effective concentration (EC50) of less than 500 nM, 250 nM, 100 nM, 50 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.25 nM, or 0.1 nM or with a EC50 within a range bounded by any of the foregoing. In some embodiments, the binding of the target domain neutralizes and / or facilitates gastrointestinal clearance of the pathogen or the pathogenic toxin. A pathogen or pathogenic toxin is “neutralized” when its ability to cause disease in an infected subject by targeting and / or interacting with the subject’s cells is decreased or eliminated. The binding may neutralize the pathogen or pathogenic toxin via any suitable mechanism, including, without limitation, via (1) physical, chemical, or biochemical modification of the pathogen or pathogenic toxin, (2) physical sequestration of the pathogen or pathogenic toxin, and / or (3) interference with the interaction of the pathogen or pathogenic toxin with the subject’s cells (e.g., via blocking of a binding site on the pathogen or pathogenic toxin).

[0052] As used herein, a “proliferative disorder” refers to a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. In some embodiments, the disorder is a proliferative disorder, and the target domain is a therapeutic agent to treat the proliferative disorder. The methods may be used to treat any proliferative disorder affecting the gastrointestinal tract. In some embodiments, the proliferative disorder is a cancer. In some embodiments, the cancer is colorectal cancer. The cancer treatment can be characterized by at least one of the following: (a) reducing, slowing or inhibiting growth of cancer and cancer cells, including slowing or inhibiting the growth of metastatic cancer cells; (b) preventing further growth of tumors; (c) reducing or preventing metastasis of cancer cells within a subject; and (d) reducing or ameliorating at least one symptom of cancer.

[0053] Methods of Detection

[0054] The present disclosure also provides methods of detecting a gastrointestinal disorder or proliferative disorder within the gastrointestinal tract. The methods comprise (a) orally administering an engineered yeast or composition comprising the engineered yeast described herein, and (b) detecting the engineered yeast, wherein the detection, location, or duration of the yeast in the gastrointestinal tract is indicative of a disorder. The detecting may comprise detection of a signal generated by the detectable tag, and may be performed using any suitable method known in the art. In some embodiments, the detecting is performed by in vivo fluorescence imaging, luminescence detection, or magnetic resonance imaging.

[0055] The methods may be used to detect any suitable gastrointestinal disorder or proliferative disorder within the gastrointestinal tract. In some embodiments, the proliferative disorder is a cancer. In some embodiments, the cancer is colorectal cancer.

[0056] Kits

[0057] The present disclosure also provides kits for preparing an engineered 5. boulardii yeast, comprising an auxotrophic S. boulardii yeast and an a-agglutinin surface display expression construct derived from S. cerevisiae. The kits may comprise any 5. boulardii yeast described herein, including, without limitation, a commercially available S. boulardii strain DBVPG 6763. In some embodiments, the auxotrophic S. boulardii yeast is a URA3 auxotroph. In some embodiments, the engineered yeast is stable in gastrointestinal conditions.

[0058] In an aspect, the expression construct encodes (i) a cloning site to allow for cloning of a target domain linked to an Aga2 domain, and an Agal domain, and (ii) promoters to allow expression of both the Agal domain and the target domain linked to the Aga2 domain. Standard cloning techniques are well known in the art. In some embodiments, the cloning site comprises a standard multiple cloning site that comprises multiple restriction enzyme sites that may also overlap and / or comprise recombination sites for recombinant cloning. In some embodiments, the cloning site is recognized by a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or an RNA-guided DNA endonuclease enzyme. In some embodiments, the kits further comprise reagents for CRISPR-Cas gene editing. In some embodiments, the expression construct is comprised within a vector. Any suitable vector disclosed herein may be used, including, without limitation plasmids, viral vectors, cosmids, and artificial chromosomes. In some embodiments, the expression construct is capable of being integrated into a chromosome of the yeast. In some embodiments, the expression construct is comprised within an integration plasmid. In some embodiments, the integration plasmid comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 5, or a nucleic acid having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 5.

[0059] In some embodiments, the target domain comprises a detectable tag. The detectable tag may be any suitable tag described herein. In some embodiments, the target domain is configured to bind to a target associated with a gastrointestinal disorder or proliferative disorder. The target domain may be configured to bind to any suitable gastrointestinal disorder or proliferative disorder described herein.

[0060] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0061] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10%> to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument, and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0062] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0063] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0064] EXEMPLARY EMBODIMENTS

[0065] Embodiment 1. An engineered Saccharomyces boulardii (S. boulardii) yeast comprising a heterologous agglutinin polynucleotide linked to a target domain polynucleotide encoding a target domain in frame to allow for production of an exogenous protein including the target domain, wherein the exogenous protein is displayed on the surface of or released from the yeast.

[0066] Embodiment 2. The engineered yeast of embodiment 1, wherein the exogenous protein comprises the target domain linked to an Aga2 domain, and the Aga2 domain is conjugated to an Agal protein via at least one disulfide bond.

[0067] Embodiment 3. The engineered yeast of embodiment 2, wherein the target domain is an antibody or a single-chain variable fragment.

[0068] Embodiment 4. The engineered yeast of embodiment 2 or 3, wherein the target domain is linked by a linker peptide to the Aga2 domain.

[0069] Embodiment s. The engineered yeast of any one of embodiments 1-4, wherein the exogenous protein further comprises a detectable tag.

[0070] Embodiment 6. The engineered yeast of embodiment 5, wherein the detectable tag is an affinity tag, an epitope tag and / or a fluorescent peptide or marker, optionally wherein the detectable tag is a His tag, a HA tag, a Myc tag, a FLAG tag, a V5 tag, a NE tag, a GST tag, a GFP tag, a RFP tag, or a fluorescent dye.

[0071] Embodiment 7. The engineered yeast of any one of embodiments 1-6, wherein the exogenous protein is configured to bind to a target associated with a gastrointestinal disorder.

[0072] Embodiment 8. The engineered yeast of any one of embodiments 1-7, wherein the engineered yeast is S. boulardii strain DBVPG 6763.

[0073] Embodiment 9. The engineered yeast of any one of embodiments 1-8, wherein the engineered yeast is auxotrophic.

[0074] Embodiment 10. The engineered yeast of embodiment 9, wherein the engineered yeast is a URA3 auxotroph.

[0075] Embodiment 11. The engineered yeast of any one of embodiments 1-10, wherein the heterologous agglutinin polynucleotide is integrated into the S. boulardii genome.

[0076] Embodiment 12. The engineered yeast of any one of embodiments 1-11, wherein the heterologous agglutinin polynucleotide is comprised within an a-agglutinin surface display construct derived from Saccharomyces cerevisiae.

[0077] Embodiment 13. The engineered yeast of any one of embodiments 1-12, wherein the engineered yeast is stable in gastrointestinal conditions. Embodiment 14. A composition comprising the engineered yeast of any one of embodiments 1-13 and a pharmaceutically acceptable carrier.

[0078] Embodiment 15. The composition of embodiment 14, wherein the composition comprises 1 million to 20 billion colony forming units of the engineered yeast.

[0079] Embodiment 16. The composition of embodiment 14 or 15, wherein the composition is formulated for oral administration.

[0080] Embodiment A method of treatment comprising administering the yeast of any one of embodiments 1-13 or the composition of any one of embodiments 14-16 to a subject having or suspected of having a gastrointestinal or proliferative disorder.

[0081] Embodiment 18. The method of embodiment 17, wherein the composition is administered orally.

[0082] Embodiment 19. The method of embodiment 17 or 18, wherein the gastrointestinal disorder is associated with a pathogen and / or a pathogenic toxin.

[0083] Embodiment 20. The method of embodiment 19, wherein the target domain binds to the pathogen or the pathogenic toxin.

[0084] Embodiment 21. The method of embodiment 20, wherein the binding neutralizes and / or facilitates gastrointestinal clearance of the pathogen or pathogenic toxin.

[0085] Embodiment 22. The method of any one of embodiments 19-21, wherein the pathogen is from genus Escherichia, Salmonella, Shigella, Enter obacter, Klebsiella, Citrobacter, Staphylococcus, Vibrio, Campylobacter, Helicobacter, Clostridium, Bacillus, Listeria, or Yersinia.

[0086] Embodiment 23. The method of any one of embodiments 19-21, wherein the pathogenic toxin is a Shiga toxin, E. coli heat-labile toxin, E. coli heat-stable toxin, Salmonella enterotoxin, C. difficile toxin A, C. difficile toxin B, a cholera toxin, a staphylococcal enterotoxin, cereulide, a B. cereus enterotoxin, listeriolysin O, a cytolethal distending toxin, or vacuolating cytotoxin A.

[0087] Embodiment 24. The method of embodiment 17 or 18, wherein the disorder is a proliferative disorder and the target domain is a therapeutic agent to treat the proliferative disorder.

[0088] Embodiment 25. The method of any one of embodiments 17-24, wherein the target domain comprises a therapeutic agent and a mucus binding agent and / or a collagen binding agent. Embodiment 26. The method of embodiment 25, wherein the mucus binding agent and / or the collagen binding agent maintains the yeast in the lower gastrointestinal tract longer than a reference yeast not including the mucus binding agent and / or the collagen binding agent.

[0089] Embodiment 27. The method of any one of embodiments 17-26, wherein the exogenous protein is delivered to the lower gastrointestinal tract of the subject.

[0090] Embodiment 28. A method of detecting a gastrointestinal disorder or proliferative disorder within the gastrointestinal tract of a subject comprising:

[0091] (a) orally administering the engineered yeast of any one of embodiments 1-13 or the composition of any one of embodiments 14-16;

[0092] (b) detecting the engineered yeast, wherein the detection, location or duration of the yeast in the gastrointestinal tract is indicative of a disorder.

[0093] Embodiment The method of embodiment 28, wherein the composition comprises 1 million to 20 billion colony forming units of the engineered yeast.

[0094] Embodiment The method of embodiment 28 or 29, wherein the gastrointestinal disorder or proliferative disorder is a cancer.

[0095] Embodiment 31. The method of embodiment 30, wherein the cancer is colorectal cancer.

[0096] Embodiment 32. The method of any one of embodiments 28-31, wherein the detecting is performed by in vivo fluorescence imaging or magnetic resonance imaging.

[0097] Embodiment 33. A kit for preparing an engineered Saccharomyces boulardii (S. boulardii) yeast comprising:

[0098] (a) an auxotrophic S. boulardii yeast; and

[0099] (b) an exogenous a-agglutinin surface display expression construct derived from Saccharomyces cerevisiae (S. cerevisiae), wherein the expression construct encodes (i) a cloning site to allow for cloning of a target domain linked to an Aga2 domain and an Agal domain and (ii) promoters to allow expression of both the Agal domain and the target domain linked to the Aga2 domain.

[0100] Embodiment 34. The kit of embodiment 33, wherein the expression construct is capable of being integrated into a chromosome of the yeast.

[0101] Embodiment 35. The kit of embodiment 33 or 34, wherein the target domain comprises a detectable tag. Embodiment 36. The kit of embodiment 35, wherein the detectable tag is an affinity tag, an epitope tag and / or a fluorescent peptide or marker, optionally wherein the detectable tag is a His tag, a HA tag, a Myc tag, a FLAG tag, a V5 tag, a NE tag, a GST tag, a GFP tag, a RFP tag, or a fluorescent dye.

[0102] Embodiment 37. The kit of any one of embodiments 33-36, wherein the target domain is configured to bind to a target associated with a gastrointestinal disorder or proliferative disorder.

[0103] Embodiment 38. The kit of any one of embodiments 33-37, wherein the auxotrophic yeast is S. boulardii strain DBVPG 6763.

[0104] Embodiment 39. The kit of any one of embodiments 33-38, wherein the auxotrophic yeast is a URA3 auxotroph.

[0105] Embodiment 40. The kit of any one of embodiments 33-39, wherein the engineered yeast is stable in gastrointestinal conditions.

[0106] EXAMPLES

[0107] Example 1 - A functional surface display system in Saccharomyces boulardii for protein absorption in simulated intestinal fluids

[0108] There is untapped potential for medical breakthroughs using engineered microbes to treat conditions such as those of the gastrointestinal tract. In this report, we explore the feasibility of established protein surface display methods to be used in the probiotic yeast Saccharomyces boulardii to display antibodies within a simulated gastrointestinal environment. We demonstrate that an auxotrophic strain of this yeast derived directly from a commercial probiotic can express functional antibodies tethered to the cell surface. We found these cell wall-tethered antibodies are able to bind their target antigen at physiological temperature and in simulated colonic fluids, mimicking the conditions in the human body where an orally ingested probiotic yeast may encounter microbially generated toxins or other biomolecules. We also demonstrate that this approach is sensitive to sub-nanomolar concentrations of antigen, providing evidence that an antibody-displaying yeast cell can potentially be effective at binding very low concentrations of target.

[0109] Introduction

[0110] Saccharomyces boulardii is a non-pathogenic yeast that has been widely used as a probiotic for the prevention and treatment of various gastrointestinal disorders such as antibiotic- associated diarrhea, inflammatory bowel disease, and Clostridium difficile infection1'2. The beneficial effects of S. boulardii are attributed to its ability to influence the intestinal microbiota, enhance the mucosal barrier function, and modulate the immune response. In addition to being a tested probiotic, it is a species of budding yeast closely related to Sacchciromyces cerevisiae. Because of this, there are a host of molecular tools and methods available for modifying S. cerevisiae that translate well into S. boulardii, making S. boulardii a prime candidate for exploring the possibility of generating a genetically modified probiotic. This includes the potential of S. boulardii as a platform for delivering therapeutic biomolecules to the gut. This has remained largely unexplored until very recently, particularly when it comes to secreting antibodies or other molecules into the gut3-8.

[0111] Surface display systems are genetic engineering tools that allow the expression of heterologous proteins on the cell surface of microorganisms such as yeast9. These systems enable the immobilization of enzymes, antibodies, and other biomolecules on the cell surface, which can facilitate their characterization and evolution10-12. Several surface display systems have been developed for S. cerevisiae, one of the most utilized species of yeast for biotechnology applications as well as food production. However, S. cerevisiae is not an ideal candidate for oral administration, as it is sensitive to gastric acid and bile salts and does not tolerate body temperature very well13. In contrast, S. boulardii is more resistant to harsh gastrointestinal conditions, grows at 37°C, and has a proven safety record as a probiotic. Therefore, utilizing a surface display system in S. boulardii may offer a way to safely and effectively generate a therapeutic outcome in the gut. Surface display of protein has recently been demonstrated in S. boulardii, providing evidence that this concept is indeed viable14'15.

[0112] Enteric diseases caused by bacteria are a global issue, and there are not adequate therapies for many of them. One solution to this may be the use of probiotic organisms that can express therapeutic molecules. One modality that, to our knowledge, has not yet been fully explored is the use of cell surface displayed neutralizing antibodies in colonic environments. In contrast to related options such as secretion of neutralizing antibodies by S. boulardii6, surface display possesses the potential advantage of a better effective affinity and removal of toxin proteins due to the avidity effect of antibodies grouped on the cell surface and the fact that this system would inherently move these captured proteins away from the site of infection as the yeast cell passes through the gastrointestinal tract (GIT). In this study, we report the application of a functional surface display system in a strain of ,S'. boulardii derived from a commercial probiotic and based on the a-agglutinin cell-surface anchoring system from S. cerevisiae. We demonstrate that this system can efficiently display genetically encoded single-chain antibodies against fibroblast activation protein (FAP) and chicken egg lysozyme and that the displayed antibodies retain their functionality in simulated colonic fluids. This study provides a proof-of-concept for an antibody surface display system in S. boulardii that is functional within simulated colonic fluids, informing future applications of antibody surface display within the gut.

[0113] Results

[0114] Generating an S. boulardii auxotroph from a probiotic strain

[0115] The long-term goal of a technology like this is to generate strains of therapeutic probiotic yeast that would be safe to ingest. Toward that end, we began our study by isolating a strain of yeast which is available commercially to be taken as a probiotic supplement. A strain of S. boulardii described as DBVPG 6763 and marketed by Pure Therapro Rx® was resuspended in water from a dried pill form and plated onto complete yeast media (YPD). This yielded colonies from which we derived the subsequent strains used in this study. Our first step was to generate an auxotrophic strain for utility. We also wanted to demonstrate that an auxotrophic probiotic strain, which may be a desirable quality in a genetically engineered therapeutic to prevent rampant proliferation in the environment, is effective at surface display. We chose to knock out the URA3 gene because this is a common and versatile selectable marker in yeast biology. This knock out strategy allows us to negatively select against cells that are prototrophic for this gene by using the uracil analogue 5-FOA, which is converted to a toxic metabolite in URA3 proficient cells. To do this, we followed a strategy16using two loxP -flanked deletion cassettes, KanMX and BleMX, containing either G418 or phleomycin resistance cassettes, respectively. This gave us a strategy for selecting for replacement of each URA3 allele sequentially via antibiotic resistance as well as a means to remove these cassettes via Cre-lox recombination. These cassettes were sequentially transformed and integrated by homologous recombination to delete about 900 bp of the URA3 open reading frame. FIG. 1A shows a table describing the primer sets used to verify cassette insertion and removal along with corresponding PCR amplicons resolved on an agarose gel. The UE primer set anneals to sequences just outside of the deleted sequence and therefore generates a unique PCR product for each genotype. The TF primer set produces a different sized band for each cassette because each cassette utilizes the Tefl promoter sequence targeted by this primer. The KN primer is specific only to the KanMX cassette insertion and produces a single band. After confirming URA3 knockout by PCR and antibiotic plating, the antibiotic cassettes were removed by recombination at the flanking loxP sites by transforming the cells with a plasmid utilizing URA3 selection and coding for the Cre recombinase17. Cells from this transformation were grown in non- selective media to remove the Cre-containing plasmid. Colonies were then screened for loss of the plasmid leading to selection of the strain used moving forward (FIG. IB).

[0116] Construction of the cell-surface antibody expression cassette

[0117] We next built an expression construct based on the conventional two-part a-agglutinin system for yeast surface display10. We designed a construct using a bidirectional TDH3(GAP)ITEF1 S. cerevisiae promoter to drive both the AGA1 gene and the gene coding for the Aga2-antibody fusion protein (FIG. 1C). We chose two single-chain antibodies to use for this investigation; a single-chain fragment variable (scFv) antibody recognizing fibroblast activation protein (FAP)18, and a camelid nanobody recognizing chicken egg-white lysozyme19. These antibody genes were fused to the 3’ end of the AGA2 gene and separated by an HA tag and a linker sequence. These cassettes were then incorporated into one of two different genomic locations, one in chromosome XI and the other in chromosome XII as described in the methods. This was verified by PCR using primer sets flanking the insertion site (FIG. ID). Strains containing either the antifibroblast activation protein cassette or the anti-lysozyme cassette are subsequently referred to as anti-FAP or anti-Lysozyme, respectively. Each plasmid is named to include the chromosomal incorporation site and the antibody for easy identification.

[0118] Strain Validation

[0119] Most studies utilize the MYA-796™ strain of S. boidardii available from ATCC. To validate that the parental strain of yeast used in this study was indeed a derivative of 5. boulardii, we sequenced the genome of the URA3S strain described above as well as the two derivative strains into which the anti-FAP expression cassette was integrated. To assess the sequencing data, the samples were aligned to the S. cerevisiae reference genome. The samples were evaluated using IGV (Integrated Genomics Viewer) and three S. boulardii genes containing consequential mutations relative to S. cerevisiae were found to be present (FIG. 2 and FIG. 6). PGM2 contains three reported mutations, two of which are silent and one of which is a nonsense mutation (G1278A) leading to a deficiency in galactose metabolism20. All three of these mutations were detected in the strain we used. Two other mutant genes are described in a study looking into increased acetic acid production in S. boulardii2. The study identified two missense mutations in the gene SDH1 (C604T, T950A). We also found these two mutations to be present in our sequencing data. They also identified a nonsense mutation in the gene WHI2 (C860G) and found this to be a heterozygous mutation in most strains of 5. boulardii they tested. Indeed, our sequencing data provides evidence that our strain is heterozygous for this mutation. It was demonstrated that these mutations were consequential and a likely hallmark difference between S. cerevisiae and S. boulardii, leading to a large increase in acetic acid production which they attribute to the ability of S. boulardii to thrive in alternative ecological niches due to its anti -microbial effect. The URA3 deletion as well as the expression cassette containing the fusion construct were also detected in the correct locations (FIG. 7). The full sequencing data generated by Oxford Nanopore Technology sequencing is available online for readers who may be interested in additional analyses.

[0120] Confirmation of cell surface antibody expression and functionality

[0121] To detect expression of the construct, we utilized a fluorescent anti-HA antibody to detect the HA tag within the Aga2-antibody fusion protein. Lysozyme or recombinant FAP conjugated to a fluorophore was used to detect antigen binding (see illustration in FIG. 3A). We initially tested cell surface expression using plasmid-based expression constructs with uracil selectable markers but ultimately decided to generate a strain with a stably integrated two-part Agal / Aga2-antibody system in the interest of simulating a more therapeutically relevant probiotic designed for use outside of media that imposes a selective pressure. To do this, we utilized the EasyClone- MarkerFree method22and validated that two of the sites tested in S. cerevisiae (XI-3 and XIL5) also work in S. boulardii. We next demonstrated that fusion protein anchoring is dependent on a disulfide linkage by treating yeast cells with two different reducing agents, DTT and PME (FIG. 3B). Treatment with either reducing agent resulted in complete loss of the anti-HA signal suggesting that the antibodies are both anchored through a disulfide bond and present on the cell surface. To further validate antibody expression, we performed a Western blot utilizing the anti- FAP strains (FIG. 3C). Although the molecular weight of the fusion protein based on the amino acid sequence is 39 kDa, it appears to resolve at an apparent molecular weight of about 50 kDa suggesting possible post-translational modification. Using flow cytometry, we were able to confirm both expression of the cell surface antibody as well as antibody-specific binding of both FAP and lysozyme in each respective strain, in both cases using strains in which the cassette is integrated into chromosome XII (FIG. 3D). This was also visualized through microscopy demonstrating co-localization of the antibody and antigen on the cell surface (FIG. 3E).

[0122] Testing the stability of YSD in simulated colonic fluids

[0123] A major question we are interested in investigating is whether genetically encoded surface displayed antibodies function in a gastrointestinal environment. We cultured samples overnight at 37°C in either tap water, yeast media (YPD), Fasted-State Simulated Colonic Fluids (FaSSCoF) or Fed-State Simulated Colonic Fluids (FeSSCoF). The next day, an aliquot of each sample was either washed and resuspended in flow cytometry buffer (see methods) or left in the overnight culture medium and stained with a fluorescent antibody against the HA epitope tag and a fluorescent antigen conjugate. To gauge viability, each sample was also stained with Sytox blue. These samples were analyzed by flow cytometry, shown in FIG. 4. The first conclusion from this experiment is that these yeast strains are viable in simulated colonic fluids for at least 18 hours (FIG. 4A). As expected, cells incubated in water or grown in YPD, a standard optimal medium for culturing yeast cells, are almost all viable. Cells cultured in simulated colonic fluids are about 40-60% viable, suggesting they are capable of surviving in colonic fluids. The second conclusion from this experiment is that these yeast cells are capable of expressing antibodies which maintain functionality and remain on the cell surface regardless of whether the cells have been incubating in water, culture media, or simulated colonic fluids (FIG. 4B). Notably, antigen binding efficiency in simulated colonic fluids appears robust, suggesting that the basic chemical components of colonic fluids do not prohibit antibody-antigen interactions. We note that there is some autofluorescence that occurs in yeast cells exposed to fasted-state colonic fluids. Still, when presented with antigen, there is an observable shift in fluorescence for the entire population indicating functional antibody expression. In each case in FIG. 4B, both live and dead cell populations are represented. Another notable observation is that even dead yeast cells (or at least those with compromised membranes permeant to Sytox dye) maintain functional antibodies on their cell surface, as greater than 85% of cells in both colonic fluid types still interact specifically with the cognate antigen compared to control cells despite almost half of those populations staining positive for Sytox stain.

[0124] Characterizing fusion protein expression and binding capacity To better understand the binding capacity of the surface display system in our strain, we wanted to characterize antigen binding and antibody display. We first determined the ECso for anti- HA binding to the expressed HA tag and FAP antigen binding to the yeast cell surface-bound antibody in an anti-FAP strain (FIG. 5A). Using flow cytometry, we determined that the anti-HA antibody (clone 2-2.2.14) binds to anti-FAP expressing cells with an ECso of 15 nM. Notably, the FAP antigen binds at an ECso of <1 nM, indicating the potential for antibody-displaying cells to specifically bind proteins at very low concentrations - a desirable characteristic for a potential therapeutic modality.

[0125] We also estimated the density of antibody expression in our strains. To do this, we used flow cytometry to generate a standard curve for PE fluorescence intensity (red-shaded Quantibrite sample, FIG. 5B). We evaluated two different strains containing the inserted fusion genes at chromosome XII using an anti-HA antibody conjugated to PE. Using this assay, we determined these cells were capable of displaying around 10,000 molecules per cell on average from the chromosomally-integrated expression cassettes (FIG. 5C). The expression density also appears to be growth phase-dependent. Cells that had time to recover in fresh media from stationay phase growth expressed about 5 times more fusion protein on the cell surface than cells from saturated cultures.

[0126] Discussion

[0127] Our goals in this study were to 1) understand the feasibility of expressing a cell surface antibody fusion protein in Saccharomyces boulardii, 2) determine if these cells could maintain viability and functional expression in simulated colonic fluids, and 3) gauge at what level of antigen concentrations these cells may be able to efficiently interact with their target. We started by generating a uracil auxotroph from a wild-type strain of Saccharomyces boulardii commercially available for use in humans for its potential therapeutic properties in the gut. We decided to move to a stable expression model whereby we integrated the Agal and Aga2-antibody genes into the genome using a CRISPR-based method. This led to strains that homogenously expressed functional cell surface antibodies against fibroblast activation protein and lysozyme. We then confirmed that the antigen binding properties persist after 18 hours in simulated colonic fluids. Additionally, about half of the population is still viable after incubating in simulated colonic fluids and even dead or dying cells still contain functional antibody. These experiments provide evidence that an antibody displaying 5. boulardii cell has the potential to be both viable and functional within a gastrointestinal environment. Lastly, we confirmed that our strains are capable of expressing approximately 10,000 antibodies per cell on the cell surface when actively proliferating and that sub-nanomolar EC50 values are achievable. The potential for high affinity antibody -target interactions will of course vary based on the antibody, but we find here that likely therapeutic levels are achievable with this model.

[0128] This study demonstrates there is exciting potential for using genetically modified probiotic organisms as a therapeutic intervention for gastrointestinal diseases. The next step is to analyze cell viability and antibody expression and functionality in an animal model to recapitulate our observations in vivo, including the use of antibodies targeting therapeutically relevant targets. There are multiple exciting routes of therapeutic molecule delivery with probiotic organisms as the research community has begun to explore in recent years. One of the greatest advantages of using genetically encoded probiotic-based therapeutics will be the ability to produce and store them very cheaply. Because the cells are the therapeutic factories themselves and can be freeze- dried and stabilized long-term at room temperature, they hold great potential to be a very low-cost and environmentally friendly (compared to drug manufacturing) means of treating some of the most rampant diseases around the world. There is also a lot of room for innovation in this growing field of therapeutic modality. For example, other groups are working on ways to enhance the effectiveness of S. boulardii within the gut through methods including surface display of proteins to enhance gut residence time23. Advances like these may have an additive effect on therapeutic efficacy in the future. We are optimistic and anticipate great potential in probiotic-based therapeutics for the treatment of a variety of gastrointestinal and microbiome-related diseases and excited to see how genetically encoded therapeutics can help to improve the treatment of disease in terms of efficiency of cost, availability, delivery, and potency.

[0129] Future work to characterize the S. boulardii functional display system will include a mammalian model, with antigen binding measured in cells that have passed through the gastrointestinal tract and into the colon. The impact of changes in transcriptional elements, environment, gene copy number, and interactions with other microorganisms on surface expression will also be explored.

[0130] Materials and Methods

[0131] Data and code availability. The whole genome sequencing FASTQ data files have been deposited at NCBI SRA under BioProject ID PRJNA1222401 Experimental model'. The strain of Saccharomyces boulardii used in this paper is diploid and derived from a commercial probiotic strain described as strain DBVPG 6763). While no official authentication process has been used to verify this, cells were sourced directly from a probiotic product. However, as a means of validating whether this strain is a strain of S. boulardii, we have sequenced the genome and verified multiple mutations that differentiate S. boulardii from S. cerevisiae. See FIG. 2 for details. The sequencing data is also available at NCBI.

[0132] Generally, cells were cultured in standard YPD medium at 30°C in glass culture tubes with rotation at 40 RPM, unless otherwise noted for specific experimental conditions. YPD medium with 2% agar was used for streaking and maintaining cells short-term.

[0133] Strain construction'. To generate a URA3 auxotroph, we used a method similar to that used in these studies6 16and described in the results section. Knockout of URA3 and the antibiotic cassettes (BleMX and KanMX) that were used to intermediately replace URA3 was verified by both PCR analysis as well as 5’FOA plating. A list of primers used in this study can be found in the Table I .

[0134] To generate the stable antibody-expressing strain of yeast, we used integration plasmids22containing homology arms for a genomic location within chromosome XI and XII, a guide RNA, and Cas9. These plasmids were obtained from Addgene as part of the EasyClone-MarkerFree kit and are as follows: pCfB2904 and pCfB2909 (integration plasmids), pCfB3045 and pCfB3O5O (gRNA plasmids), and pCfB2312 (Cas9 plasmid). Integration plasmids were linearized by PCR for use in the transformations. Transformation was accomplished using the Frozen-EZ Yeast Transformation II Kit #T2001 according to the instructions and with extended incubation times suggested in the manual. YPD was purchased from Sunrise Science. Agar was purchased from Difco. A list of plasmids and primers utilized for this work can be found in Table 1.

[0135] Media The following media products were used for growing yeast: YPD (Sunrise Science #1875- 500), Agar (Difco #214530), Synthetic media components (YNB+Nitrogen - Sunrise Science #1501-250, L-Tryptophan - Sunrise Science # 1987-010, SC-Trp-Ura - Sunrise Science #1316- 030, Glucose (for synthetic media) - Sunrise Science #19O7-1KG)

[0136] PCR. PCR for cloning was performed using Clonamp HiFi PCR Premix (Takara #639298). For genotyping, Phire plant direct PCR master mix was used (Thermo Scientific #F160). 1.2% agarose (Fisher Scientific #BP160) gels were used for the genotyping analysis. DNA ladders used were either Invitrogen 100 bp DNA ladder (#15628019) or O’GeneRuler Express DNA ladder (#SM1551). Sequencing-. Primer-less Oxford Nanopore sequencing was performed by Plasmidsaurus Inc. for the whole genome sequencing analysis. Each sample of S. boulardii was grown overnight in YPD, then diluted and grown for 5 more hours to 10,000,000 cells / ml, pelleted, and resuspended in DNA / RNA Shield (Zymo Research #R1100) and sent to Plasmidsaurus for processing and sequencing.

[0137] After processing, base-calling was performed by Plasmidsaurus and FASTQ files were returned. These were sorted and aligned to the S. cerevisiae S288C genome (GCF_000146045.2_R64_genomic acquired from NCBI) using EPI2ME software to generate BAM files. BAM files were visualized in IGV (Integrated Genomics Viewer) to analyze the reads and compare the 3 samples to each other and to the S. cerevisiae strain S288C. FASTQ files are deposited at the NCBI Sequence Read Archive under accession number PRJNA1222401.

[0138] Cartoons and schematics'. FIG. 3A cartoon was generated using BioRender - BioRender.com / g64y958. DNA schematics were generated using SnapGene software version 7.2.1. IGV (Integrated Genomics Viewer) was used to generate figures derived from whole genome sequencing data.

[0139] Western blot. Cells were grown overnight in 5 ml cultures. Cultures were diluted 1: 10 in 10 ml YPD and grown for 3 hours the next morning before pelleting at 500 xg for 3 minutes at room temperature and washing with phosphate-buffered saline twice. Samples were resuspended in 100 pl of 10 mM Tris pH 7.5, 0.6% SDS, 1 mM EDTA, protease and phosphatase inhibitors (Thermo Scientific #78442), and glass beads (Sigma #G8772) and vortexed at max speed for 5 minutes. Samples were then centrifuged for 3 minutes at 5,000 xg and the supernatant collected. LDS sample buffer was added to each sample (Life Technologies #B0007) and heated at 85°C for 5 minutes. Samples were run at 100V on a Bis-Tris 4-12% gel (Invitrogen #NW04122BOX), then transferred to a PVDF membrane using a Pierce Power Station semi-dry transfer. The blot was blocked with 2.5% BSA (bovine serum albumin) in TBST pH 7.6 (tris-buffered saline with 0.1% tween-20). Anti-HA (Invitrogen #26183-0650, RRID AB 2533053) and anti-Gapdh (Invitrogen #MA5-15738, RRID AB_10977387) were each (separately) diluted 1:500 and 1 : 1000 respectively and incubated with the blot at room temperature for 1.5 hours. An anti-mouse Alexa Fluor 647 (Invitrogen #A21235, RRID AB_2535804) antibody diluted at 1 :5000 was incubated with the blot for 1 hour at room temperature for detection of the anti-Gapdh antibody. A GE ImageQuant LAS 4000 imager was used to generate exposures via fluorescence in the Cy5 channel. The uncropped exposures can be found in FIG. 8.

[0140] Antigen binding assays and flow cytometry

[0141] Cell growth'. Antigen binding and cell-surface antibody detection assays were performed to characterize cell-surface antibody expression and functionality in vitro. In these assays, S. boulardii yeast cells were initially grown overnight at 30°C in YPD. The next morning, they were diluted 50-fold into fresh YPD medium and grown for 4 more hours at 30°C before assaying 100 pl aliquots (approximately 7xl06cells / ml concentration). Cultures used in experiments performed in simulated colonic fluids were additionally cultured as follows. For both simulated colonic fluids and tap water, 4 ml cultures were inoculated at 2 million cells / ml and grown overnight at 37°C for approximately 18 hours. The YPD cultures were inoculated at 5,000 cells / ml so that they would be not be in stationary phase by the time of the experiment. Incubation was performed in glass culture tubes on a rotator. Simulated colonic fluids were prepared from components from Biorelevant according to the instructions available on the website within a pH of + / - 0.1. FaSSCoF was prepared at pH 7.8 and FeSSCoF was at pH 6.0. (FaSSCoF COFAS01 and FeSSCoF # COFES01).

[0142] Binding Assays'. 100 pl aliquots were taken from each sample culture and pelleted at 500 xg for 3 minutes at room temperature to collect cells. If not assayed in overnight culture buffer, cells were washed twice with flow cytometry buffer (Invitrogen #00-4222-26) and then resuspended in the designated buffer. All subsequent centrifugation steps performed between washes were done at 500 xg for 3 minutes at 4°C. Each sample was then simultaneously incubated with 1 pl / 0.2 pg of anti -HA antibody conjugated to PE (Biolegend #901518, RRID AB 2629623) and lpl / 100 nM concentration of Fibroblast activation protein (Biolegend #768904) or Lysozyme (Sigma #A55O3) conjugated to iFluor 647 (AAT Bioquest #1963) in 100 pl volume for 30 minutes at 4°C. Samples were then washed twice, stained with 1 pM Sytox blue (Thermo #S34857) (1 / 1000 dilution of stock) (Life Technologies #S34857) when assessing viability, and resuspended in flow cytometry buffer before acquiring on an Attune NxT flow cytometer. The gating strategy used in all flow cytometry experiments was to first gate on singlets using a forward scatter height by area plot. Forward by side scatter height was then used to gate the primary cell population. Sytox blue was captured in a 440 / 50 channel off a violet laser, PE was captured in a 585 / 16 channel off a yellow laser, and iFluor 647 was captured in a 670 / 14 channel off a red laser. Release of cell-surface antibodies with reducing agents. Yeast cells were grown in YPD for 4 hours after reinoculation of overnight culture, then pelleted by centrifugation at 500 x g for 3 minutes and resuspended in either 10 mM Dithiothreitol in 25 mM Tris pH 7.5, 1% Betamercaptoethanol in 25 mM Tris pH 7.5, or 25 mM Tris pH 7.5 for 30 minutes at room temperature. These samples were then washed with water followed by flow cytometry buffer (Invitrogen #00- 4222-26). Samples were incubated with anti-HA antibody conjugated to DyLight 650 (Invitrogen #26183) for 15 minutes at room temperature, washed and evaluated by flow cytometry on an Attune NxT flow cytometer. Data was plotted using Attune NxT software.

[0143] Microscopy. Samples were prepared the same as for the flow cytometry antigen binding experiments before fixing to slides. After treating the cells with anti-HA antibody and fluorescently conjugated FAP protein, yeast cells were fixed in 4% formaldehyde solution and spotted onto glass slides in 3 pl drops. Samples were mounted with coverslips using ProLong Glass antifade mountant (Invitrogen #P36982) and visualized using a Zeiss LSM 880 confocal laser microscope with Airyscan.

[0144] Affinity Measurements'. ECso determinations were made as follows. 2,500 yeast cells in log phase growth were transferred in 45 pl of flow cytometry buffer to each well of a 96-well plate pre-blocked with flow cytometry buffer. FAP conjugated to Dylight 405 (Invitrogen #46401) or anti-HA antibody (Invitrogen #26183-0650, RRID AB_2533053) were added in 5 pl aliquots (from serial dilutions) to each sample, mixed by pipetting, and incubated on a shaker for 1 hour at room temperature. The samples were washed twice with flow cytometry buffer and acquired on an Attune NxT flow cytometer. Calculations were performed in GraphPad Prism version 10.3.0 for Windows using median fluorescence intensity values and a 4 parameter least squares fit. The control strain lacking expression of the fusion protein was used to subtract any background fluorescence due to off-target antibody binding or cellular autofluorescence.

[0145] Antibody Binding Capacity (ABC) measurement'. ABC was determined using BD Quantibrite Beads (#340495) on an Attune NxT flow cytometer. BD Quantibrite Beads contains a mixture of beads with known amounts of conjugated PE (phycoerythrin). This was used to generate a standard curve and approximate the number of PE conjugated anti-HA antibodies (Biolegend #901518, RRID AB_2629623) bound per cell using flow cytometry MFI values. Calculations assume 1.22 PE molecules per antibody as reported by the manufacturer (Biolegend). At a saturating concentration of antibodies, this value should then be equal to the number of HA-tag containing Aga2-scFv proteins expressed per cell since there is a single HA sequence expressed in each surface fusion protein. Antibodies were incubated with cells at 4°C for 45 minutes, then washed with flow cytometry buffer before acquiring on the flow cytometer. Flow cytometry buffer- resuspended Quantibrite Beads were also acquired during the same run. Calculations were performed as described in the Quantibrite product information and using lot-specific values. For this experiment, cells were grown overnight to saturation in YPD. The next morning, new cultures were inoculated 1 / 50. For each time point (0, 2, 4, 6 hours), 100 pl of each culture was collected and stained with antibody using the same protocol described in the “Binding assays” section.

[0146] Quantification and Statistical Analysis'. Statistical details of experiments can be found in figure legends where relevant. GraphPad Prism was used to plot FIG. 5A. Microsoft Excel was used for all other graphs and analyses.

[0147] Table 1: Resources

[0148] References

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Claims

CLAIMSWhat is claimed:

1. An engineered Saccharomyces boulardii (S. boulardii) yeast comprising a heterologous agglutinin polynucleotide linked to a target domain polynucleotide encoding a target domain in frame to allow for production of an exogenous protein including the target domain, wherein the exogenous protein is displayed on the surface of or released from the yeast.

2. The engineered yeast of claim 1, wherein the exogenous protein comprises the target domain linked to an Aga2 domain, and the Aga2 domain is conjugated to an Agal protein via at least one disulfide bond.

3. The engineered yeast of claim 2, wherein the target domain is an antibody or a singlechain variable fragment.

4. The engineered yeast of claim 2, wherein the target domain is linked by a linker peptide to the Aga2 domain.

5. The engineered yeast of claim 1, wherein the exogenous protein further comprises a detectable tag.

6. The engineered yeast of claim 5, wherein the detectable tag is an affinity tag, an epitope tag and / or a fluorescent peptide or marker, optionally wherein the detectable tag is a His tag, a HA tag, a Myc tag, a FLAG tag, a V5 tag, a NE tag, a GST tag, a GFP tag, a RFP tag, or a fluorescent dye.

7. The engineered yeast of claim 1, wherein the exogenous protein is configured to bind to a target associated with a gastrointestinal disorder.

8. The engineered yeast of claim 1, wherein the engineered yeast is S. boulardii strain DBVPG 6763.

9. The engineered yeast of claim 1, wherein the engineered yeast is auxotrophic.

10. The engineered yeast of claim 9, wherein the engineered yeast is a URA3 auxotroph.

11. The engineered yeast of claim 1, wherein the heterologous agglutinin polynucleotide is integrated into the S. boulardii genome.

12. The engineered yeast of claim 1, wherein the heterologous agglutinin polynucleotide is comprised within an a-agglutinin surface display construct derived from Saccharomyces cerevisiae.

13. The engineered yeast of claim 1, wherein the engineered yeast is stable in gastrointestinal conditions.

14. A composition comprising the engineered yeast of any one of claims 1-13 and a pharmaceutically acceptable carrier.

15. The composition of claim 14, wherein the composition comprises 1 million to 20 billion colony forming units of the engineered yeast.

16. The composition of claim 14 or 15, wherein the composition is formulated for oral administration.

17. A method of treatment comprising administering the yeast of any one of claims 1-13 or the composition of any one of claims 14-16 to a subject having or suspected of having a gastrointestinal or proliferative disorder.

18. The method of claim 17, wherein the composition is administered orally.

19. The method of claim 17 or 18, wherein the gastrointestinal disorder is associated with a pathogen and / or a pathogenic toxin.

20. The method of claim 19, wherein the target domain binds to the pathogen or the pathogenic toxin.

21. The method of claim 20, wherein the binding neutralizes and / or facilitates gastrointestinal clearance of the pathogen or pathogenic toxin.

22. The method of any one of claims 19-21, wherein the pathogen is from genus Escherichia Salmonella, Shigella, Enterobacter, Klebsiella, Citrobacter, Staphylococcus, Vibrio, Campylobacter, Helicobacter, Clostridium, Bacillus, Listeria, or Yersinia.

23. The method of any one of claims 19-21, wherein the pathogenic toxin is a Shiga toxin, E. coli heat-labile toxin, E. coli heat-stable toxin, Salmonella enterotoxin, C. difficile toxin A, C. difficile toxin B, a cholera toxin, a staphylococcal enterotoxin, cereulide, a B. cereus enterotoxin, listeriolysin O, a cytolethal distending toxin, or vacuolating cytotoxin A.

24. The method of claim 17 or 18, wherein the disorder is a proliferative disorder and the target domain is a therapeutic agent to treat the proliferative disorder.

25. The method of any one of claims 17-24, wherein the target domain comprises a therapeutic agent and a mucus binding agent and / or a collagen binding agent.

26. The method of claim 25, wherein the mucus binding agent and / or the collagen binding agent maintains the yeast in the lower gastrointestinal tract longer than a reference yeast not including the mucus binding agent and / or the collagen binding agent.

27. The method of any one of claims 17-26, wherein the exogenous protein is delivered to the lower gastrointestinal tract of the subject.

28. A method of detecting a gastrointestinal disorder or proliferative disorder within the gastrointestinal tract of a subject comprising:(a) orally administering the engineered yeast of any one of claims 1-13 or the composition of any one of claims 14-16;(b) detecting the engineered yeast, wherein the detection, location or duration of the yeast in the gastrointestinal tract is indicative of a disorder.

29. The method of claim 28, wherein the composition comprises 1 million to 20 billion colony forming units of the engineered yeast.

30. The method of claim 28 or 29, wherein the gastrointestinal disorder or proliferative disorder is a cancer.31 . The method of claim 30, wherein the cancer is colorectal cancer.

32. The method of any one of claims 28-31, wherein the detecting is performed by in vivo fluorescence imaging or magnetic resonance imaging.

33. A kit for preparing an engineered Saccharomyces boulardii (S. boulardii) yeast comprising:(a) an auxotrophic S. boulardii yeast; and(b) an exogenous a-agglutinin surface display expression construct derived from Saccharomyces cerevisiae (S. cerevisiae), wherein the expression construct encodes (i) a cloning site to allow for cloning of a target domain linked to an Aga2 domain and an Agal domain and (ii) promoters to allow expression of both the Agal domain and the target domain linked to the Aga2 domain.

34. The kit of claim 33, wherein the expression construct is capable of being integrated into a chromosome of the yeast.

35. The kit of claim 33 or 34, wherein the target domain comprises a detectable tag.

36. The kit of claim 35, wherein the detectable tag is an affinity tag, an epitope tag and / or a fluorescent peptide or marker, optionally wherein the detectable tag is a His tag, a HA tag, a Myc tag, a FLAG tag, a V5 tag, a NE tag, a GST tag, a GFP tag, a RFP tag, or a fluorescent dye.

37. The kit of any one of claims 33-36, wherein the target domain is configured to bind to a target associated with a gastrointestinal disorder or proliferative disorder.

38. The kit of any one of claims 33-37, wherein the auxotrophic yeast is 5. boulardii strain DBVPG 6763.

39. The kit of any one of claims 33-38, wherein the auxotrophic yeast is a URA3 auxotroph.

40. The kit of any one of claims 33-39, wherein the engineered yeast is stable in gastrointestinal conditions.

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