Compositions and uses for engineered therapeutic microorganisms and associated receptors

Abstract

Described herein are microbial probiotics that secrete anti-inflammatory proteins, such as IL-2, IL-10, or the CD39-like eATPase apyrase, in response to extracellular ATP (eATP), a metabolite produced in the microenvironment of detected inflamed tissue, e.g., via an engineered mammalian P2Y2 receptor. Accordingly, provided herein are isolated Saccharomyces cells (or a plurality of cells, e.g., a population of such cells) that have been engineered to express one, two, or all three exogenous proteins selected from the following: (i) a mammalian P2Y purinergic receptor 2 (P2Y2) protein, preferably human P2Y2; (ii) a mammalian Gα, preferably Gα; i3 a mutant Gpa1 protein comprising at least five C-terminal residues derived from Gpa1, wherein the mutant Gpa1 protein couples the P2Y2 protein to the yeast mating pathway; and (iii) an anti-inflammatory protein.

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of U.S. Provisional Patent Application No. 62 / 891,603, filed August 26, 2019, the entire contents of which are incorporated herein by reference.

[0002] Described herein are microbial probiotics that secrete anti-inflammatory proteins, such as IL-2, IL-10, or the CD39-like eATP-degrading enzyme apyrase, in response to extracellular ATP (eATP), a metabolite produced in the microenvironment of detected inflamed tissue, e.g., via engineered mammalian P2Y purinergic receptor 2 (P2Y2). [Background technology]

[0003] Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract, including Crohn's disease and ulcerative colitis (1). Most available IBD treatments systemically suppress the immune system, increasing the risk of infections and some types of cancer (2). In addition, many patients with IBD do not respond to treatment or show a loss of clinical response over time (2). Therefore, there is a need for novel therapeutic approaches for IBD.

[0004] Microbes regulate immune processes associated with the pathogenesis of multiple human diseases, including IBD (3-5). For example, IBD-associated single nucleotide polymorphisms promote alterations in the gut microbiota that result in reduced production of anti-inflammatory microbial metabolites (6). IBD-associated genetic polymorphisms also modulate responsiveness to anti-inflammatory microbial metabolites (7). The role of the microbiome in disease development, and in particular the anti-inflammatory effects of certain commensal microorganisms, supports the use of probiotic-based approaches for the treatment of IBD (8-10). However, treatments based solely on the endogenous anti-inflammatory properties of unmanipulated probiotics may be ineffective in modulating ongoing intestinal inflammation (10). Summary of the Invention

[0005] The development of therapeutic probiotics is a major area of IBD research. Previous attempts have used plasmid-based engineered probiotics that express therapeutic proteins in an uncontrolled manner, requiring constant selection pressure and posing the risk of horizontal transmission to other bacteria (80, 81). To overcome these limitations, we applied directed evolution and CRISPR-Cas9 to engineer microorganisms with genetic circuits that produce anti-inflammatory drugs in response to eATP levels, delivering a dynamic probiotic-based anti-inflammatory response in inflamed tissue microenvironments. Provided herein are engineered therapeutic microorganisms designed to detect pathogenic gastrointestinal (GI) inflammation and dynamically respond by delivering therapeutic proteins, and methods for their use.

[0006] Accordingly, provided herein is an isolated Saccharomyces cell (or a plurality of cells, e.g., a population of such cells) that has been engineered to express one, two, or all three exogenous proteins selected from the following: (i) a mammalian P2Y purinergic receptor 2 (P2Y2) protein, preferably human P2Y2; (ii) a mammalian Gα, preferably Gα i3and (iii) an anti-inflammatory protein, optionally in a mammalian, preferably human, cell line, wherein the anti-inflammatory protein is expressed under the control of a promoter activated downstream of P2Y2 activation, optionally a mating-responsive promoter, and wherein the isolated Saccharomyces cells secrete the anti-inflammatory protein in the presence of extracellular adenosine triphosphate (eATP). Preferably, the anti-inflammatory protein is secreted in the presence of eATP at pro-inflammatory concentrations (about 100 micromolar to high millimolar). Preferably, the anti-inflammatory protein is secreted in an eATP concentration-dependent manner, with higher eATP concentrations resulting in greater secretion of the anti-inflammatory protein within the dynamic range of the engineered P2Y2 receptor.

[0007] In some embodiments, the Saccharomyces cells have been engineered to reduce or eliminate expression of one or more endogenous proteins selected from the group consisting of: (i) yeast GPCR, e.g., the α-factor pheromone receptor STE2 (NP_116627.2); (ii) a negative regulator of pathway function, the GTPase-activating protein SST2 (NP_013557.1); (iii) the cell cycle regulator cyclin-dependent protein serine / threonine kinase inhibitory protein FAR1 (NP_012378.1); and (iv) the yeast Gα protein guanine nucleotide-binding protein subunit αGPA1 (NP_011868.1).

[0008] In some embodiments, the anti-inflammatory protein includes a yeast-derived leader peptide that directs the protein to be secreted, and optionally lacks any signal or leader sequence endogenous to the anti-inflammatory protein.

[0009] In some embodiments, the anti-inflammatory protein comprises apyrase, interleukin 10 (IL-10), IL-2, IL-27, IL-22, or IFN-β.

[0010] In some embodiments, at least one of the P2Y2 protein, mutant Gpa1, or anti-inflammatory protein is expressed from a sequence that is codon-optimized for expression in a Saccharomyces cell.

[0011] In some embodiments, P2Y2 comprises one or more mutations that increase expression of an anti-inflammatory protein. In some embodiments, the mutations are at residues surrounding the ligand binding pocket (optionally, A76 2.47 , N116 3.35 , C119 3.38 , L162 4.54 , Q165 4.57 ), and / or residues on the intracellular-facing side of the receptor (optionally F58 1.57 , L59 1.58 , C60 1.59 , A229 ICL3 , K240 6.31 , F307 7.54 , G310 C末端 In some embodiments, the one or more mutations are at residue F58 1.57 , N116 3.35 , F307 7.54 , and / or Q165 4.57In some embodiments, the one or more mutations include F58C, Q165H, F307S, and / or N116S. In some embodiments, the mutation includes a mutation at N116. In some embodiments, the mutation includes a mutation at N116 in combination with a mutation at F58 or F307. In some embodiments, the mutation includes a mutation N116S, optionally in combination with a mutation F58I or F307S. In some embodiments, P2Y2 further includes a mutation at L59 and / or C119. In some embodiments, the additional mutation includes L59I and / or C119S.

[0012] In some embodiments, the promoter activated downstream of P2Y2 activation is a mating-responsive promoter, for example, pFUS1 or pFIG1.

[0013] In some embodiments, expression of the anti-inflammatory protein is driven by a synthetic transcription factor containing a pheromone response domain and a DNA binding domain that binds to a non-yeast DNA operator sequence upstream of the sequence encoding the anti-inflammatory protein.

[0014] In some embodiments, the isolated Saccharomyces cell is S. cerevisiae or S. boulardii.

[0015] Also provided herein are compositions comprising an isolated Saccharomyces cell as described herein and, optionally, a physiologically acceptable carrier. In some embodiments, the composition is in a solid form for oral administration, such as a tablet, pill, capsule, soft gelatin capsule, dragee, orodispersing / orodispersing tablets, or effervescent tablet.

[0016] In some embodiments, the composition is in the form of a liquid for oral administration, for example, a drinking solution.

[0017] In some embodiments, the composition is a nutritional composition, optionally comprising liquid or solid food, feed, or drinking water.

[0018] In some embodiments, the nutritional composition is selected from beverages, optionally smoothies or fermented beverages, flavored beverages, yogurt, drinking yogurt, set yogurt, fruit and / or vegetable juices or concentrates thereof, fruit and vegetable juice powders, reconstituted fruit products, powders, malt or soy or grain based beverages, breakfast cereals such as muesli flakes, spreads, meal replacements, confectionery, chocolates, gels, ice cream, cereal, fruit and / or chocolate bars, energy bars, snack bars, food bars, sauces, dips, and sports supplements, including dairy and non-dairy based sports supplements.

[0019] Also provided herein is a method for reducing inflammation in a subject, comprising administering to the subject an effective amount of an isolated Saccharomyces cell or composition as described herein.Further provided are isolated Saccharomyces cells and compositions for use in methods for reducing inflammation in a subject.In some embodiments, the subject has or is at risk of developing inflammatory bowel disease (IBD).

[0020] Additionally, residues around the ligand binding pocket (optionally A76 2.47 , N116 3.35 , C119 3.38 , L162 4.54 , Q165 4.57 ), and / or residues on the intracellular-facing side of the receptor (optionally F58 1.57 , L59 1.58 , C60 1.59 , A229 ICL3, K240 6.31 , F307 7.54 , G310 C末端 Provided herein are engineered mammalian P2Y purinergic receptor 2 (P2Y2) proteins comprising one or more mutations in the nucleotide sequence (SEQ ID NO: 1) of the P2Y2 receptor.

[0021] One or more mutations at residue F58 1.57 , N116 3.35 , F307 7.54 , and / or Q165 4.57 31. The engineered mammalian P2Y2 of claim 30, wherein the P2Y2 is in

[0022] 32. The engineered mammalian P2Y2 of claim 31 , wherein the one or more mutations comprise F58C, Q165H, F307S, and / or N116S.

[0023] 31. The engineered mammalian P2Y2 of claim 30, wherein the mutation comprises a mutation at N116.

[0024] 34. The engineered mammalian P2Y2 of claim 33, wherein the mutation comprises a mutation at N116 in combination with a mutation at F58 or F307.

[0025] 35. The engineered mammalian P2Y2 of claim 34, wherein the mutation comprises the mutation N116S, optionally in combination with the mutation F58I or F307S.

[0026] 36. The engineered mammalian P2Y2 of any one of claims 30 to 35, wherein the P2Y2 further comprises a mutation at L59 and / or C119.

[0027] 37. The engineered mammalian P2Y2 of claim 36, wherein the additional mutations include L59I and / or C119S.

[0028] 38. An isolated nucleic acid sequence encoding the engineered mammalian P2Y2 of any one of claims 30 to 37.

[0029] 38. A host cell comprising the isolated nucleic acid sequence of claim 35, and optionally expressing the engineered mammalian P2Y2 of any one of claims 30 to 37.

[0030] 40. The host cell of claim 39, wherein the cell is a Saccharomyces cell and the isolated nucleic acid sequence is codon-optimized for expression in the Saccharomyces cell.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0032] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. [Brief explanation of the drawings]

[0033] [Figure 1-1]Figures 1A-D show directed evolution of the human P2Y purinergic receptor 2 (P2Y2). (A) Human P2Y2 receptor activation was functionally coupled to expression of the fluorescent reporter protein, mCherry, using the mating-responsive promoter pFUS1. Modifications to the mating pathway included knockout of the negative regulator Sst2 and the gene encoding Far1, which arrests cell growth in the wild-type mating pathway. The chimeric Gα protein (Gpa1-Gαi3) contains the five C-terminal amino acids of mammalian Gαi3. (B) The engineered mating pathway responds to UTP using various yeast strains harboring wild-type (WT) human P2Y2 receptor and the Gpa1-Gα chimera, including BS019 (G14), BS020 (Gq), and BS016 (Gi3). After 6 h of incubation with the indicated concentrations of UTP, activation of the mating pathway was monitored by quantitating mCherry fluorescence by flow cytometry. Data points represent the mean of two colonies. Error bars represent SEM. *P<0.05 vs. 0 μM UTP. (C) Cells expressing human WT P2Y2 receptor were treated with UTP and ATP, and mCherry fluorescence was quantified by flow cytometry. Data points represent the mean of six colonies for ATP and the mean of three colonies for UTP. Error bars represent SEM. (D) A plasmid library of human P2Y2 receptor mutants generated by error-prone PCR was transformed into the Gpa1-Gαi3 mCherry receptor strain. Cells were treated with 100 μM ATP and selected for highly activated mutants (top 1% of mCherry fluorescence) using fluorescence-activated cell sorting. Individual yeast colonies were then screened to confirm the desired phenotype and sequenced. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 1-3] This is a continuation of Figure 1-1. [Figure 2-1]Figures 2A-B show the increased responsiveness to eATP of human P2Y2 receptor mutants generated by directed evolution. (A) Randomly selected yeast colonies were incubated with the indicated ligands for 6 hours, and mCherry fluorescence was quantified. Responses were normalized to WT human P2Y2 receptor activated with 100 μM ATP, with the y-axis representing the fold increase above the WT response. Ten yeast colonies were selected for detailed characterization (purple boxes), and their responses to 100 μM ATP and 100 μM UTP are shown in the inset. (B) Multiple mutations in the human P2Y2 receptor increased the sensitivity and maximal response to eATP and eUTP. mCherry fluorescence is expressed as a percentage of the maximal WT response to eATP. Mutants are grouped based on the position of the mutant residue. Data points represent the average of six colonies for eATP and three colonies for eUTP, each transformed with a plasmid encoding the indicated human P2Y2 receptor mutant. Error bars represent SEM. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 2-3] This is a continuation of Figure 2-1. [Figure 3-1]Figures 3A-H show the characterization of human P2Y2 receptor mutants. (A) Residues mutated in the human P2Y2 receptor after directed evolution. The top 10 mutant human P2Y2 receptors with enhanced ATP sensitivity were classified based on the location of the mutant residues: helix 1, helix 7, and the transmembrane region. ATP docked in the putative binding pocket is shown in light gray. Residues F58, Q165, and F307 were mutated in eight of the top 10 mutants. Modeling was performed using MODELLER 9.18 based on the P2Y1 receptor structure (4XNW.pdb). (B) Expression of C-terminally GFP-tagged human P2Y2 receptor mutants in yeast was quantified by flow cytometry. Average GFP values were normalized to WT P2Y2 expression. Data are the average of at least three colonies, and error bars represent standard deviation. *P<0.05 vs. WT. (C) Representative images of GFP-tagged endogenous yeast STE2 GPCR and human P2Y2 receptor mutants examined by confocal microscopy. Scale bar represents 5 μM. (D-G) Combinations of human P2Y2 receptor mutations generated by directed evolution reveal novel GPCR features. (D) Combining the N116S mutation with either F58I or F307S resulted in the most active human P2Y2 receptor. The F307S mutation confers constitutive activity, improves sensitivity to eATP, and enhances response to eATP. (E) Non-additive effects contribute to the increased activity of the human P2Y2 receptor TM-2 mutant. The L59I and C119S mutations alone confer a modest increase in sensitivity to ATP. Their combined effect is lower than that detected in the TM-2 mutant, indicating a non-additive change. (F) Separate analysis of each mutation in the H1-1 mutant identified F58C as the primary mutation affecting activity. K240N did not contribute to the detected increase in activity. (G) The TM-1 mutant harbors silent mutations in addition to the Q165H mutation. Silent mutations contribute to increased ATP sensitivity, including when they are combined with the F58I mutation. mCherry fluorescence is expressed as a percentage of the maximum wild-type response to ATP.Data points represent the average of at least three colonies, each transformed with a plasmid encoding the indicated P2Y2 mutant. Error bars represent SEM. (H) P2Y2 residue F58 was mutated to all other amino acids, and the dose response to eATP was assessed using the Gpa1-Gαi3 mCherry reporter strain. mCherry fluorescence is expressed as a percentage of the maximal wild-type response to ATP. Data points represent the average of at least three colonies, each transformed with a plasmid encoding the indicated P2Y2 mutant. Error bars represent SEM. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 3-3] This is a continuation of Figure 3-1. [Figure 3-4] This is a continuation of Figure 3-1. [Figure 3-5] This is a continuation of Figure 3-1. [Figure 3-6] This is a continuation of Figure 3-1. [Figure 3-7] This is a continuation of Figure 3-1. [Figure 4-1]Figures 4A-F show eATP-responsive secretion of ATPase by engineered yeast. (A) Sequence alignment of apyrase genes. Human ENTPD1 (CD39), potato apyrase (RROP1), and wheat apyrase (TUAP1) were aligned using MUSCLE in MEGA6 Alignment Explorer. (B) Therapeutic response element. (C) Cell lysates from yeast strains constitutively expressing potato apyrase (RROP1) or wheat apyrase (TUAP1), or a vector-free yeast strain expressing no apyrase. Bands correspond to the C-terminal HA tag on apyrase. RROP1 is predicted to appear at 48 kDa without the N-terminal α-factor signal peptide, or 57 kDa with the signal peptide. TUAP1 is predicted to appear at 46 kDa without the signal peptide and 55 kDa with the signal peptide. The arrow indicates the cytoplasmic protein Pgk1, used as a loading control. (D) Five microliters of supernatant from strains constitutively secreting potato apyrase (RROP1) or wheat apyrase (TUAP1) was incubated with 50 μM ATP for 30 minutes in a 50 μL total reaction volume, and residual ATP was quantified; the parent yeast strain that does not express apyrase (CB008) served as a negative control. Each figure represents three biological replicates. Error bars represent standard deviations; *P<0.001. (E) Activation of the engineered human P2Y2 receptor was functionally coupled to expression of the RROP1 apyrase using the mating-responsive promoter pFUS1. Upon receptor activation by eATP, apyrase is expressed and secreted thanks to a signal peptide that facilitates secretion by yeast. Secreted apyrase dephosphorylates extracellular ATP to ADP and AMP, which then shuts off the gene circuit. (F) Engineered yeast strains harboring the P2Y2 / RROP1 gene circuit were incubated with the indicated concentrations of ATP for 16 hours. ATPase activity was quantified by incubating 5 μL culture supernatant with 50 μM ATP in a 50 μL reaction for 30 minutes, after which time the remaining ATP was measured. ATPase unit = 1 μmol of ATP to ADP per minute.From left to right: AP-P4 (WT); APTM-3 (N116S); APH1-1 (F58C C60Y G310A); APH1-3 (F58I); APTM-2 (L59I C119S); APTM-1 (Q165H); APH7-1 (K240N F307S); and constitutive apyrase (BS029). "Constitutive" refers to a yeast strain expressing RROP1 under the control of the strong constitutive pTDH3 promoter. Data are the average of three biological replicates performed on separate days, and error bars represent the standard deviation. *P<0.05 vs. WT response at the same ATP concentration. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 4-3] This is a continuation of Figure 4-1. [Figure 4-4] This is a continuation of Figure 4-1. [Figure 4-5] This is a continuation of Figure 4-1. [Figure 4-6] This is a continuation of Figure 4-1. [Figure 5-1]Figures 5A-K show that eATP-responsive synthetic yeast ameliorates TNBS-induced colitis. (A) mCherry-positive yeast (% of total GFP yeast) were quantified by flow cytometry in the fecal contents of specific intestinal segments 2 hours after oral gavage with the ATP-inducible TM-3 yeast strain (left) or BS035 constitutive (right). ATP levels were measured in the same segments of the intestine. (B) Changes in body weight after rectal administration of TNBS. Statistical significance between groups was assessed by two-way ANOVA followed by Tukey's multiple comparison post-hoc test; ***P<0.05; *P<0.05; ns=not significant; n=10). (C) Colon lengths of mice (n=4) from the experimental groups shown in (B). (D) Hematoxylin and eosin staining at 20x (top) and 40x (bottom) magnification. Representative intestinal segments from each group are shown. Open arrowheads: immune cells infiltrating the mucosa with structural destruction. Black arrows: immune cell infiltration in the submucosa. Black brackets: edematous submucosa. Scale bar = 100 μm. (E) Histomorphological disease scores of mice from the group as in (B), with higher scores indicating greater severity of tissue destruction (n = 4). (F) RNA-Seq analysis of colon samples from mice in the experimental group shown in (B). Heatmap of differentially expressed genes. (G) Foxp3+ T regulatory cells in mesenteric lymph nodes in the experimental group shown in (B) (n = 3). (H) Foxp3, Ifng, and Il17 mRNA expression determined by qPCR in colon tissue of samples from the group as in (B) (n = 3). (I) Changes in body weight over the course of DSS-induced colitis in mice treated with probiotic yeast as in (B). Statistical significance between groups was assessed by two-way ANOVA followed by Tukey's multiple comparison post-hoc test, **P<0.01, *P<0.05; ns=not significant; (n=12). (J) Gene expression in colon samples from yeast-treated mice as determined by NanoString 21 days after the start of DSS administration. Heatmap of differentially expressed genes. Data are representative of two independent experiments with pooled samples from n=3 mice per group.(K) Nos2, Ccl2, and Il1b mRNA expression (n=3) determined by qPCR in RNA extracted from colon tissue from mice from groups as in (J). ***P<0.01, **P<0.01, *P<0.05; ns=not significant, as determined by one-way ANOVA followed by post hoc Tukey or Sidak test for selected multiple comparisons. Data are representative of three independent experiments. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-1. [Figure 5-4] This is a continuation of Figure 5-1. [Figure 5-5] This is a continuation of Figure 5-1. [Figure 5-6] This is a continuation of Figure 5-1. [Figure 5-7] This is a continuation of Figure 5-1. [Figure 5-8] This is a continuation of Figure 5-1. [Figure 5-9] This is a continuation of Figure 5-1. [Figure 6-1]Figures 6A-H show that eATP-responsive synthetic yeast probiotics limit fibrosis and dysbiosis. (A) Masson Trichrome staining for fibrosis, 20x (top) and 40x (bottom) magnification. Fibrotic areas are stained and highlighted with white arrows. Scale bar = 100 μm. (B) Histology fibrosis scores (n = 4) of mice from the group from (L). (C-H) High-throughput gene sequencing analysis of microbial 16S rRNA genes performed by MiSeq on fecal samples. (C) Alpha diversity of the fecal microbiome. The Shannon index, which compares differences in alpha diversity, was calculated at the highest sequencing depth (4000 pb). *P < 0.05; ns = not significant, as determined by Kruskal-Wallis nonparametric ANOVA test. (D-E) Beta diversity. (D) Principal coordinate analysis (PCoA) based on the unweighted UniFrac metric. (E) Unweighted UniFrac distance relative to the ethanol control group. *P<0.05 Permanova analysis. (F) Relative abundance of bacteria classified at the family level of taxonomy. (G) Relative abundance of Lachnospiraceae and its genus Roseburia. (H) LEfSe p<0.05 for the APTM-3 vs. CB008 comparison. Each cladogram represents all taxa detected at >0.1%, presented from the kingdom phylogenetic level to the genus level. Light gray circles represent taxa that are present but not abundant. Dark gray circles are abundant in APTM-3, and striped circles are abundant in CB008. **P<0.01; *P<0.05; ns=not significant, as determined by one-way ANOVA followed by a post-hoc Tukey's test. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 6-3] This is a continuation of Figure 6-1. [Figure 6-4] This is a continuation of Figure 6-1. [Figure 6-5] This is a continuation of Figure 6-1. [Figure 6-6] This is a continuation of Figure 6-1. [Figure 6-7] This is a continuation of Figure 6-1. [Figure 6-8] This is a continuation of Figure 6-1. [Figure 7] Figure 7A-B shows the response of the engineered mating pathway to eATP over time. (A, B) Yeast from strain BS016 transformed with plasmid pRS316 pTDH3 P2Y2 (WT human P2Y2 receptor) was incubated with 100 μM ATP in 300 μL (A) or 5 mL (B) of SD-URA medium, and mCherry fluorescence was quantified (each from two individual colonies; error bars represent standard deviation). [Figure 8] Figure 8 shows a diagram illustrating the strategy for directed evolution of the human P2Y2 receptor. During each FACS sort, approximately the top 1% of mCherry fluorescence was collected. "Recovered" refers to the number of yeast colonies obtained after plating sorted cells on selective medium. [Figure 9] Figures 9A-B show ATP concentrations in yeast supernatants. (A) The slopes were not statistically different. (B) To estimate the amount of active apyrase secreted by yeast, 50 μM ATP was incubated with 5 μL of supernatant from a culture of strain CB008 in a 50 μL reaction volume at 30°C for 30 min with the indicated concentrations of commercial apyrase, and the remaining ATP was quantified. When 31.3 pM of commercial apyrase was added, no apyrase activity was observed. [Figure 10-1]Figures 10A-D show that synthetic yeast probiotics are viable in the mouse intestine. (A) Colony-forming units per mg of feces collected 6 hours after oral gavage of mice with either the CB008 KG, BS029 KG, or APTM-3 KG yeast strains. (B) Relative ATP levels in specific segments of the intestine of naive and TNBS-induced mice. (C) mCherry-positive yeast (% of total GFP yeast) measured by flow cytometry in the fecal contents of specific segments of the intestine 2 hours after oral gavage of naive mice with the ATP-inducible TM3 strain, TM-3 KG (right). ATP levels were measured in the same segments of the intestine. (D) mCherry-positive yeast (% of total GFP yeast) quantified by flow cytometry in the fecal contents of specific segments of the intestine 2 hours after oral gavage of TM-3 KG or P4 KG (WT) yeast strains. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 10-3] This is a continuation of Figure 10-1. [Figure 11-1] Figure 11 shows the plasmid pCAS AarI. A custom multiple cloning site was inserted into the XmaI and BglII sites in the pCAS plasmid (SEQ ID NO: 22) obtained from AddGene (112). The image was generated using the CLC sequence viewer. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 12]Figure 12 illustrates how ATP-responsive therapeutic microbes regulate purinergic signaling during inflammation. Chronic inflammation is characterized by upregulated extracellular ATP (eATP), reaching levels >100 μM surrounding inflamed tissue (Bours, MJ, Dagnelie, PC, Giuliani, AL, Wesselius, A. & Di Virgilio, F. P2 receptors and extracellular ATP: a novel homeostatic pathway in inflammation. Frontiers in bioscience 3, 1443-1456 (2011); Di Virgilio, F., Pinton, P. & Falzoni, S. Assessing Extracellular ATP as Danger Signal In Vivo: The pmeLuc System. Methods in molecular biology 1417, 115-129 (2016)). eATP induces pro-inflammatory responses from various immune and epithelial cells in the intestine, primarily mediated through the P2X7 receptor (Kurashima, Y., Kiyono, H. & Kunisawa, J. Pathophysiological role of extracellular purinergic mediators in the control of intestinal inflammation. Mediators of inflammation 2015, 427125 (2015)). Activation of P2X7 promotes caspase-1 expression, leading to the maturation of inflammatory cytokines and the opening of pannexin-1 (Panx1) channels, promoting the release of additional ATP (Cekic, C. & Linden, J. Purinergic regulation of the immune system. Nature reviews. Immunology 16, 177-192 (2016)).To prevent eATP accumulation, the ectonucleotidases CD39 and CD73 degrade ATP into ADP, AMP, and ultimately adenosine (Cekic, C. & Linden, (2016)). A2A, A2B, and A3 receptors are GPCRs primarily expressed by immune cells, and their activation by adenosine leads to an anti-inflammatory response (Cekic, C. & Linden, (2016)). The eATP-responsive therapeutic microorganism of the present invention dynamically regulates these existing immunoregulatory pathways. After introduction into the GI tract, yeast cells can sense upregulated eATP via engineered P2Y2 receptors expressed on their surface. P2Y2 activates a rewired mating pathway and secretes apyrase or mouse IL-10 in an eATP concentration-dependent manner. Apyrase functions to directly degrade eATP, helping to generate anti-inflammatory adenosine while simultaneously blocking P2Y2 activation signals. IL-10 acts on the IL-10 R1 / R2 receptor, resulting in downregulation of many pro-inflammatory genes, including the NLRP3 inflammasome and caspases (Gurung, P. et al. Chronic TLR Stimulation Controls NLRP3 Inflammasome Activation through IL-10 Mediated Regulation of NLRP3 Expression and Caspase-8 Activation. Sci Rep 5, 14488 (2015); Zhang, J., Fu, S., Sun, S., Li, Z. & Guo, B. Inflammasome activation has an important role in the development of spontaneous colitis. Mucosal Immunol 7, 1139-1150 (2014)) (Paul, G., Khare, V. & Gasche, C. Inflamed gut mucosa: downstream of interleukin-10. Eur J Clin Invest 42, 95-109) (2012)). DETAILED DESCRIPTION OF THE INVENTION

[0034] The confluence of efficient genetic engineering (11, 12) and advanced synthetic gene circuit design (13, 14) has paved the way for the engineering of increasingly complex microorganisms (15-18). Indeed, recent advances in synthetic biology have enabled the engineering of probiotics to deliver therapeutic proteins in response to disease-related signals (19-22). One such signal relevant to IBD is extracellular adenosine triphosphate (eATP), which, when released by activated immune cells and commensal bacteria, signals through purinergic receptors to trigger proinflammatory cytokine production, boost effector T cell activation, suppress regulatory T cell responses, and promote enteric neuronal apoptosis, among other biological responses thought to contribute to IBD pathology (23-27). eATP signaling is limited by membrane-bound ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, also known as CD39), which hydrolyzes eATP to AMP, which is then metabolized by CD73 to immunosuppressive adenosine. CD39 limits eATP-driven proinflammatory responses while simultaneously boosting the differentiation, stability, and function of regulatory T cells (26). Further support for the physiological role of eATP and CD39 in regulating intestinal inflammation is provided by reports of dysregulation of purinergic signaling in IBD patients due to increased eATP production and / or its reduced hydrolysis (25, 28).

[0035] Genetic polymorphisms that decrease CD39 expression have been linked to Crohn's disease (68). CD39 on Tregs suppresses effector T cell generation and function in experimental and human IBD (26-28, 69). Indeed, increased CD39 levels have been associated with disease remission induced by blocking antibodies against TNFα in IBD patients (70). Conversely, eATP-driven purinergic signaling promotes inflammation through multiple mechanisms, including modulation of antigen-presenting cells (71), boosting effector T cell activation (23, 72), and reducing the function and stability of regulatory T cells (26, 27, 73). eATP also limits the production of immunoglobulin A (74), which protects the intestinal barrier and promotes the engraftment of anti-inflammatory commensal bacteria (75, 76). In addition, eATP also acts on non-immune cells, promoting IBD pathogenesis by triggering apoptosis of enteric neurons (24). Therefore, blocking eATP-driven signaling is an attractive therapeutic approach for IBD.

[0036] Depletion of eATP using apyrase has been shown to attenuate intestinal inflammation (23). These anti-inflammatory effects of apyrase likely involve both eATP depletion through the conversion of eATP to AMP and the generation of immunosuppressive adenosine from AMP (29). Adenosine is a cytotoxic hormone (A). 2A It inhibits T cell activation via adenosine receptors (29). Indeed, we recently reported that CD39-driven adenosine production suppresses tumor-specific T cells in glioblastoma (77). Therefore, modulating the eATP / adenosine balance is a promising approach for treating inflammation. However, clinical application of this approach requires appropriate methods for therapeutic administration and an inducible system that modulates the eATP / adenosine balance where and when needed to minimize undesirable side effects, such as immunosuppression and fibrosis (26, 28, 77) and gut microbiome dysregulation (29, 30).

[0037] Using directed evolution (33) and synthetic biology (34) approaches, we exploited the modular architecture of the S. cerevisiae mating pathway to engineer a strain of this yeast to express an engineered human G protein-coupled receptor (GPCR) that is activated by proinflammatory signals to trigger the secretion of therapeutic proteins. GPCRs function as biological sensors for detecting a wide variety of signals, including those indicative of disease (Marinissen, MJ & Gutkind, JS G-protein-coupled receptors and signaling networks: emerging paradigms. Trends in pharmacological sciences 22, 368-376 (2001)). This ability makes GPCRs useful components of synthetic genetic circuits that trigger programmed responses to specific disease cues (Heng, B.C., Aubel, D. & Fussenegger, M. G protein-coupled receptors revisited: therapeutic applications inspired by synthetic biology. Annu Rev Pharmacol Toxicol 54, 227-249 (2014)). The S. cerevisiae mating pathway provides a well-characterized model for GPCR signaling that can be rewired to accommodate activation by human GPCRs (Ladds, G., Goddard, A. & Davey, J. Functional analysis of heterologous GPCR signaling pathways in yeast. Trends Biotechnol 23, 367-373 (2005)).Elevated extracellular adenosine triphosphate (ATP) is a major pro-inflammatory signal (Bours, MJ, Dagnelie, PC, Giuliani, AL, Wesselius, A. & Di Virgilio, F. P2 receptors and extracellular ATP: a novel homeostatic pathway in inflammation. Frontiers in bioscience 3, 1443-1456 (2011)), is increased over 100-fold in the IBD intestine (>100 μM) (Kurashima, Y., Kiyono, H. & Kunisawa, J. Pathophysiological role of extracellular purinergic mediators in the control of intestinal inflammation. Mediators of inflammation 2015, 427125 (2015)), and is specifically detected by the purinergic family of GPCRs (Burnstock, G. & Boeynaems, JM. Purinergic signaling and immune cells. Purinergic signaling 10, 529-564 (2014)). The enzyme apyrase directly degrades ATP, converting it to the immunosuppressant adenosine (Cekic, C. & Linden, J. Purinergic regulation of the immune system. Nature reviews. Immunology 16, 177-192 (2016)), and apyrase has been shown to reduce GI inflammation in animal models of IBD (Wan, P. et al. Extracellular ATP mediates inflammatory responses in colitis via P2 x 7 receptor signaling. Sci Rep 6, 19108 (2016)).The anti-inflammatory cytokine interleukin-10 (IL-10) is important for limiting inflammatory responses in the intestine (Paul, G., Khare, V. & Gasche, C. Inflamed gut mucosa: downstream of interleukin-10. Eur J Clin Invest 42, 95-109 (2012)). Microorganisms have been engineered to constitutively secrete IL-10 rather than in response to pro-inflammatory signals (Braat, H. et al. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin Gastroenterol Hepatol 4, 754-759 (2006); Rottiers, P., Vandenbroucke, K. & Iserentant, D., Vol. EP1931762(B1). (ed. EP Office) 1-26 (Actogenix NV, Belgium; 2012)), and are showing promise for treating IBD in phase I clinical trials (Braat, H. et al. (2006)).

[0038] Described herein are microbial probiotics that secrete anti-inflammatory proteins, such as IL-2, IL-10, or the CD39-like eATP-degrading enzyme apyrase, which respond to the metabolic product eATP produced in the microenvironment of detected inflammatory tissues, depleting pro-inflammatory eATP and promoting the production of immunosuppressive adenosine, for example, via engineered human P2Y2 receptors. These engineered apyrase-expressing yeast suppressed experimental intestinal inflammation in mice and reduced intestinal fibrosis and dysbiosis. Specific molecular pathways involved in purinergic signaling during inflammation are outlined in Figure 12; without wishing to be bound by theory, Figure 12 also illustrates how engineered microbes may modulate these pathways to dynamically treat inflammation in the GI tract.

[0039] These data demonstrate that controlled eATP depletion using a yeast probiotic engineered to produce apyrase in response to eATP sensing minimizes fibrosis induction. Furthermore, the use of an inducible engineered yeast strain that modulates purinergic signaling also allowed for the restoration of a healthy microbiome, minimizing dysbiosis and other human disorders thought to contribute to IBD pathology (3, 4).

[0040] Engineered microorganisms The inherent modularity of signaling pathways (78) allows for manipulation with exogenous proteins (79). Saccharomyces species have long been known for use in food, and certain Saccharomyces species have also been used as safe probiotics with engineered genetic circuits to drive regulated expression of proteins in response to stimuli of interest (15, 31, 32). In some embodiments, the engineered microorganism is produced in S. cerevisiae. S. boulardii is more commonly used as a probiotic than S. cerevisiae (89, 90), and genetic tools for engineering S. boulardii are available (91, 92). Thus, although S. cerevisiae is exemplified herein, the inducible system described herein can also be established using other microorganisms, including S. boulardii.

[0041] In some embodiments, the microorganism is generated by modifying the genome of a parent microorganism, e.g., a Saccharomyces species, e.g., S. cerevisiae. The modifications can include, but are not limited to, the introduction of the following proteins into the yeast genome: (i) an engineered P2Y2 containing up to three mutations that make it more responsive to eATP, e.g., under the control of a constitutive promoter (pTDH3); (ii) a promoter that couples P2Y2 to the yeast mating pathway, e.g., a mammalian Gα (Gα i3 ) mutant Gpa1 proteins containing the five C-terminal residues of potato apyrase or interleukin 10 (IL-10) containing a yeast-derived leader peptide that directs the apyrase to be secreted, regulated by a promoter downstream of GPCR activation, e.g., from the Fus1 gene. Modifications can also include (but are not limited to) deletion of one or more of the following endogenous yeast proteins from the genome: (i) the native yeast GPCR mating pathway receptor Ste2 (e.g., the α-factor pheromone receptor STE2 (NP_116627.2) to avoid pathway activation by the native ligand), (ii) the negative regulator of pathway function Sst2 (e.g., the negative regulator of pathway function, GTPase-activating protein SST2 (NP_013557.1) to increase pathway response when activated by P2Y2), (iii) the cell cycle regulator Far1 (e.g., the cell cycle regulator cyclin-dependent protein serine / threonine kinase inhibitory protein FAR1 (NP_012378.1) to avoid cell cycle arrest due to mating pathway activation), and (iv) the yeast Gα protein Gpa1 (e.g., the yeast Gα protein guanine nucleotide-binding protein subunit αGPA1 (NP_011868.1) to avoid competition for binding with other pathway components).

[0042] Thus, the method involves the generation of mutant Gα proteins in which the five C-terminal amino acids of Gpa1 (KIGII) are replaced with the five C-terminal amino acids from a designated mammalian Gα protein (Brown et al., Yeast. 2000 Jan 15;16(1):11-22. 2000) (e.g., chimeric yeast Gpa1-human Gα i3 These approaches may include introducing a GPCR P2Y2 (e.g., a mutant P2Y2, optionally codon-optimized for yeast expression), P2Y2 (e.g., a mutant P2Y2, optionally codon-optimized for yeast expression), and an anti-inflammatory molecule, such as apyrase or interleukin-10 (IL-10), regulated by a promoter activated downstream of P2Y2 activation (e.g., a mating pathway-responsive promoter). As shown herein, engineered variants of the GPCR P2Y2 responded to concentrations of eATP (approximately 100 micromolar to high millimolar concentrations) indicative of inflammation. In addition, in response to P2Y2 activation, apyrase or IL-10 was secreted by the engineered yeast strain in an ATP concentration-dependent manner, with apyrase functioning to degrade extracellular ATP. Finally, in a mouse model of IBD, treatment with an engineered yeast strain secreting apyrase directly improved disease prognosis and reduced pro-inflammatory cytokine production. The engineered yeast described herein can contain, for example, an autoregulatory P2Y2-RROP1 gene circuit responsive to pro-inflammatory eATP, which is itself hydrolyzed by a secreted apyrase encoded by RROP1 to dynamically regulate the eATP / adenosine balance in a time- and location-specific manner.

[0043] Exogenous sequences can be introduced into the microorganism using molecular biology methods known in the art. In some embodiments, the engineered genetic circuit is integrated into the yeast genome, e.g., using CRISPR-mediated integration, to avoid the use of antibiotic selection markers while maintaining uracil auxotrophy for biocontainment consistent with Food and Drug Administration (FDA) guidelines for live biologic organisms (Docket No. FDA-2010-D-0500). S. cerevisiae strains are present in healthy microbiomes, are reduced during IBD (82-84), and have been associated with physiological training of the immune system (85-88).

[0044] P2Y purinergic receptor 2 (P2Y2) The P2Y2 receptor is the purinergic GPCR most sensitive to eATP and has previously been functionally linked to the S. cerevisiae mating pathway (Junger, W. G. Immune cell regulation by autocrine purinergic signaling. Nature reviews. Immunology 11, 201-212 (2011); Brown, A. J. et al. Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast / mammalian G protein alpha-subunit chimeras. Yeast 16, 11-22 (2000)). The method can include the use of yeast engineered to express a G protein-coupled receptor (GPCR) activated by pro-inflammatory signals, e.g., a P2Y2 GPCR, e.g., human P2Y2.

[0045] An exemplary reference sequence for human P2Y2 protein is provided in GenBank as NP_002555.4. Exemplary reference sequences encoding human P2Y2 protein are provided in GenBank as NM_176072.3 (variant 1); NM_002564.4 (variant 2); and NM_176071.3 (variant 3). Transcript variants 1, 2, and 3 encode the same protein. The DNA sequence of human P2Y2 used in the exemplary engineered yeast strains presented herein is codon-optimized for expression in yeast, with up to 2%, 5%, 10%, 15%, or 20% of the amino acids optionally including or in addition to the mutations described herein, for example, with respect to the protein sequence as shown in NP_002555.4 (NP_002555.4).

[0046] In some embodiments, engineered human P2Y2 is used in which mutations modulate the response to physiological levels of eATP, i.e., by increasing G protein signaling and the expression of anti-inflammatory proteins. In some embodiments, mutations are made to residues around the ligand binding pocket (A76 2.47 , N116 3.35 , C119 3.38 , L162 4.54 , Q165 4.57 ), or residues located on the intracellular-facing side of the receptor (F58 1.57 , L59 1.58 , C60 1.59 , A229 ICL3 , K240 6.31 , F307 7.54 , G310 C末端 In some embodiments, P2Y2 is at the residue that contributes most to increased eATP sensitivity (i.e., F58 1.57 , N116 3.35 , F307 7.54 , and Q165 4.57), e.g., one or more mutations at residues F58 (e.g., F58C), Q165 (e.g., Q165H), and F307 (e.g., F307S). In some embodiments, the mutations include a mutation at N116, e.g., N116S, optionally in combination with a mutation at either F58, e.g., F58I, or a mutation at F307, e.g., F307S. In some embodiments, P2Y2 includes a mutation at L59, e.g., L59I, and / or a mutation at C119, e.g., C119S. In addition to the specific mutations described herein, mutations to other amino acids can also be used, e.g., F58 can be changed to any other amino acid. (Numbering corresponds to NP_002555.4 - SEQ ID NO: 13)

[0047] anti-inflammatory agents The microorganisms described herein are engineered to express one or more anti-inflammatory agents. Exemplary anti-inflammatory agents include apyrase, interleukin-10 (IL-10), IL-2, IL-27, IL-22, and IFN-β. The anti-inflammatory agent is placed under the control of a promoter that is triggered by the binding of eATP to the GPCR P2Y2, which (without wishing to be bound by theory) causes G protein-mediated triggering of the MAP kinase cascade and expression of the anti-inflammatory agent. Exemplary promoters include pFUS1 (defined as the 1636 bp immediately upstream of the Fus1 start codon; Gene ID 850330, GenBank Accession No. NC_001135.5, range 71803-73341), or pFIG1 (defined as the 500 bp immediately upstream of the Fig1 start codon; Gene ID 852328, GenBank Accession No. NC_001134.8, range 316968-317864). Alternatively, one can use synthetic transcription factors containing pheromone-responsive and DNA-binding domains paired with non-yeast DNA operator sequences upstream of anti-inflammatory genes, similar to those described by Mukherjee et al., ACS Synth. Biol. 2015, 4, 12, 1261-1269 (2015) and Shaw et al. Cell. 177(3): 782-796.e27 (Apr 2019).

[0048] Apyrase (RROP) In mouse models of IBD and chronic inflammation, intraperitoneal injection of apyrase reduces T cell activation, blocks pro-inflammatory cytokine production, and alleviates colitis (Wan, P. et al. Extracellular ATP mediates inflammatory responses in colitis via P2 x 7 receptor signaling. Sci Rep 6, 19108 (2016); Atarashi, K. et al. ATP drives lamina propria T(H)17 cell differentiation. Nature 455, 808-812 (2008); Cauwels, A., Rogge, E., Vandendriessche, B., Shiva, S. & Brouckaert, P. Extracellular ATP drives systemic inflammation, tissue damage, and mortality. Cell death & disease 5, e1102 (2014)). Apyrase breaks down pro-inflammatory ATP and helps convert it into the anti-inflammatory signal, adenosine (Cekic, C. & Linden, J. Purinergic regulation of the immune system. Nature reviews. Immunology 16, 177-192 (2016)).

[0049] Apyrase (RROP1; GenBank accession U58597.1) isolated from potato (S. tuberosum) has the highest reported ATPase activity (115). The BlastPhyMe tool was used for genome mining for homologous genes using RROP1 as the initial input sequence (116). Apyrase from wild einkorn wheat (Triticum urartu) (designated "TUAP1" and used in the examples described herein) was ultimately selected because it conserves domains known to be required for apyrase function (Knowles, Purinergic Signal. 2011 Mar;7(1):21-45) and based on a previous report of wheat apyrase activity (Komoszynski Comp Biochem Physiol B Biochem Mol Biol. 1996 Mar;113(3):581-91; see GenBank accession KD039156.1). The DNA sequences of S. tuberosum (RROP1) and Triticum urartu (TUAP1) used in the exemplary engineered yeast strains presented herein were codon-optimized for expression in yeast (see below). In addition, the endogenous apyrase N-terminal signal peptide (e.g., the first 30 nucleotides of U58597.1 or the first 18 amino acids of KD039156.1) can be replaced with a yeast secretion signal, such as the MFα1 signal peptide (the first 85 or 89 amino acids of NP_015137.1, depending on whether a Ste13 cleavage site is desired). Other signal sequences, such as the signal sequence from prepro-α-factor (see, e.g., Wittke et al., Mol Biol Cell. 2002 Jul; 13(7): 2223-2232; Microb Cell Fact. 2014; 13: 125), or the BGL2 signal peptide (or an artificial BGL2 pre-Val 7Alternatively, a variant (see Achstetter et al., Gene 110(1): 25-21, 2 January 1992), or an AGA2 or EXG1 signal peptide sequence (see Mori et al., J. Biosci. Bioeng. 2015; 120(5):518-525), or an engineered peptide sequence not found in nature (see Rakestraw et al., Biotechnol. Bioeng. 2009; 103(6):1192-1201) can be used.

[0050] Interleukin 10 (IL-10) IL-10 acts to downregulate pro-inflammatory genes and is required for proper control of inflammation (Paul, G., Khare, V. & Gasche, C. Inflamed gut mucosa: downstream of interleukin-10. Eur J Clin Invest 42, 95-109 (2012)). Delivery of IL-10 has been investigated as a treatment for IBD, but its efficacy may be limited by the low concentration it reaches in the gut (Marlow, GJ, van Gent, D. & Ferguson, LR Why interleukin-10 supplementation does not work in Crohn's disease patients. World J Gastroenterol 19, 3931-3941 (2013)).

[0051] Exemplary reference sequences for human IL-10 protein are provided in GenBank as NP_000563.1 (interleukin-10 isoform 1 precursor) and for mouse IL-10 (mIL-10) as NP_034678.1 (interleukin-10 precursor); exemplary DNA reference sequences encoding these two are provided in GenBank as NM_000572.3 and NM_010548.2, respectively. The mIL-10 DNA sequence used in the exemplary engineered yeast strains presented herein was codon-optimized for expression in yeast. The endogenous IL-10 N-terminal signal peptide (the first 21 amino acids of NP_034678.1) can be replaced by a yeast secretion signal, such as the MFα1 signal peptide (the first 85 or 89 amino acids of NP_015137.1, depending on whether a Ste13 cleavage site is desired). Other signal sequences, such as the signal sequence from prepro-α-factor (see, e.g., Wittke et al., Mol Biol Cell. 2002 Jul; 13(7): 2223-2232; Microb Cell Fact. 2014; 13: 125), or the BGL2 signal peptide (or an artificial BGL2 pre-Val 7 Alternatively, a nucleotide sequence such as a nucleotide sequence variant (see Achstetter et al., Gene 110(1): 25-21, 2 January 1992), or an AGA2 or EXG1 signal peptide sequence (see Mori et al., J. Biosci. Bioeng. 2015; 120(5):518-525), or an engineered peptide sequence not found in nature (see Rakestraw et al., Biotechnol. Bioeng. 2009; 103(6):1192-1201) can be used. See also WO2007039586.

[0052] Interleukin-2 (IL-2) Low-dose IL-2 has been shown to expand Tregs and ameliorate disease in a humanized mouse model of experimental colitis. Goettel et al., Cell Mol Gastroenterol Hepatol. 2019; 8(2): 193-195.

[0053] An exemplary reference sequence for the human IL-2 protein is provided in GenBank as NP_000563.1; an exemplary human reference sequence encoding IL2 is provided as NM_000586.4, optionally including a yeast secretion signal as described above.

[0054] IL-27 An exemplary reference sequence for the human IL-27 protein is provided in GenBank as NP_663634.2; an exemplary human reference sequence encoding IL-27 is provided as NM_145659.3, optionally including a yeast secretion signal as described above. IL-27 therapy has been proposed as a treatment for IBD; see Andrews et al., Inflamm Bowel Dis. 2016 Sep; 22(9): 2255-2264.

[0055] IL-22 An exemplary reference sequence for the human IL-27 protein is provided in GenBank as NP_065386.1; an exemplary human reference sequence encoding IL-27 is provided as NM_020525.5, optionally including a yeast secretion signal as described above. IL-22 therapy has been proposed as a treatment for IBD; see Li et al., World J Gastroenterol. 2014 Dec 28; 20(48): 18177-18188.

[0056] Interferon-β1 (IFN-β) An exemplary reference sequence for the human IL-27 protein is provided in GenBank as NP_002167.1; an exemplary human reference sequence encoding IL-27 is provided as NM_002176.4, optionally including a yeast secretion signal as described above. Interferon beta-1a is in clinical trials for IBD, e.g., in ulcerative colitis; see, e.g., Nikolaus et al., Gut. 2003 Sep; 52(9): 1286-1290.

[0057] Codon optimization and variants In addition, the nucleic acid sequences used in the present methods and compositions are preferably codon-optimized for expression in a selected expression system, such as S. cerevisiae. To optimize expression in non-mammalian cells, codon optimization specific to the selected host organism can be used. For example, in embodiments where S. cerevisiae is used as the host organism, the following Table A (source: kazusa.or.jp) can be used to select codons:

[0058] [Table 1]

[0059] In some embodiments, the method includes a variant of the reference sequence as described herein. Thus, in some embodiments, the sequence can be at least 60%, 70%, 80%, 90%, or 100% identical to the reference sequence, or at least 80%, 85%, 90%, 95%, or 99% identical; For example, the sequence can include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations in addition to the mutations described herein, as long as these additional mutations do not significantly reduce the relevant activity of the protein (for example, as shown in Figure 12, for P2Y2, the ability to sense eATP and trigger the expression and secretion of anti-inflammatory drugs; for apyrase, the ability to decompose eATP; for IL-10, the ability to down-regulate inflammatory genes, etc.). To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The length of a reference sequence aligned for comparison purposes is typically at least 80% of the length of the reference sequence, and in some embodiments, at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.In another embodiment, the percent identity of two amino acid sequences can be evaluated as a function of the conservation of amino acid residues within the same family of amino acids (e.g., positively charged, negatively charged, polar, and uncharged, hydrophobic) at corresponding positions in both amino acid sequences (e.g., the presence of an alanine residue in place of a valine residue at a particular position in both sequences indicates a high level of conservation, while the presence of an arginine residue in place of an aspartic acid residue at a particular position in both sequences indicates a low level of conservation).

[0060] For purposes of the present invention, comparison of sequences and determination of percent identity between two sequences may be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0061] Treatment method The gut microbiome plays a central role in health and disease (67). Based on the multiple functions performed by the microbiome, the use of engineered probiotics is considered an attractive therapeutic approach for inflammatory diseases, among other human disorders. The engineered microorganisms described herein can be used, for example, in the treatment and prevention of inflammatory conditions, e.g., by administering an effective amount of the engineered microorganism to the GI tract of a patient, e.g., by oral ingestion of a composition comprising an engineered microorganism as described herein sufficient to reduce inflammation and treat the inflammatory condition, or to reduce the risk of or delay the onset of the inflammatory condition.

[0062] The microorganisms can be used, for example, in the treatment and prevention of inflammatory conditions, such as inflammatory bowel conditions, including inflammatory bowel disease (IBD), by administering the engineered microorganisms to a patient's GI tract, for example, by oral ingestion of a composition containing the engineered microorganisms. IBD can include Crohn's disease; ulcerative colitis (UC); microscopic colitis; diverticulosis-associated colitis; collagenous colitis; lymphocytic colitis; and Behçet's disease. The microorganisms can be used, for example, in the treatment and prevention of graft-versus-host disease (GVHD) or after anti-tumor therapy (e.g., chemotherapy, radiation therapy, and checkpoint inhibitors, all of which induce GI inflammation). The microorganisms can be used, for example, in the treatment and prevention of GI inflammation.

[0063] eATP promotes intestinal inflammation in intestinal conditions, including inflammatory bowel disease (IBD), as well as other diseases beyond IBD, such as graft-versus-host disease and radiation therapy-induced abdominal fibrosis (93, 94). Furthermore, the gut microbiome regulates inflammation in distal body sites, such as the central nervous system (95-97). Thus, the present method can be used for the treatment and / or prevention of inflammatory disorders that target other tissues beyond the digestive system, e.g., to reduce systemic inflammation.

[0064] Generally, the methods involve administering an effective amount of an engineered microorganism as described herein to a subject in need of, or determined to be in need of, such treatment. The methods can involve administering the microorganism as often as needed to reduce inflammation, e.g., once or twice daily, e.g., 1, 2, 3, 4, 5, 6, or 7 days (e.g., daily) per week; and administration can be continued for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more, or indefinitely.

[0065] As used in this context, "treating" means ameliorating at least one symptom of an inflammation-associated disorder (e.g., reducing the severity or frequency of the symptom); administration of a therapeutically effective amount of an engineered microorganism as described herein can result in a reduction in one or more symptoms of an inflammation-associated disorder. As noted above, in some embodiments, the disorder is IBD. For example, Crohn's disease often results in frequent diarrhea; occasional constipation; abdominal pain; fever; bloody stool; fatigue; skin disorders; joint pain; malnutrition; weight loss; and / or fistulas. UC often results in abdominal pain; loose stools; bloody stools; bowel urgency; fatigue; loss of appetite; weight loss; and / or malnutrition. Administration of a therapeutically effective amount of an engineered microorganism as described herein can result in a reduction in any one or more of these symptoms. Administration of a prophylactically effective amount of an engineered microorganism as described herein can result in a reduced risk or delayed onset of an inflammation-associated disorder. Subjects with inflammation-associated disorders can be identified by those skilled in the art, for example, using imaging methods such as colonoscopy or CT scans. In some embodiments, subjects treated using the methods described herein include those at risk for developing an inflammation-related disorder, e.g., those at higher risk than the general population as a result of genetics / family history, age, race, diet, or other risk factors. See also WO2007039586.

[0066] composition Compositions comprising engineered microorganisms are provided herein. Preferably, the compositions are formulated for oral administration of the microorganism and include carriers or excipients that are physiologically acceptable, i.e., non-toxic and do not affect the activity of the engineered microorganism.

[0067] In some embodiments, the composition is a solid form, such as a tablet, pill, capsule, soft gelatin capsule, dragee, orally disintegrating / orally disintegrating tablet, effervescent tablet, or other solid, hi some embodiments, the composition is in the form of a liquid, such as a drinking solution.

[0068] Oral compositions generally contain an inert diluent or an edible carrier. For oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, or cornstarch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavor.

[0069] In some embodiments, the composition is a nutritional composition, including liquid or solid food, feed or drinking water.In some embodiments, the composition is food, such as dairy and non-dairy based drinks, plant or animal-based milk products (such as almond, cashew, soybean or oat milk; or cow's, goat's or sheep's milk), powdered milk, reconstituted milk, fermented milk, smoothie or fermented drink (produced by fermenting sugar-containing medium), flavored drinks, yogurt, drinking yogurt, solid yogurt, fruit and / or vegetable juice or its concentrate, fruit and vegetable juice powder, reconstituted fruit product, powder, or malt or soybean or grain-based drinks, and beverages, including dairy and non-dairy based sports supplements; or breakfast cereals such as muesli flakes, spreads, meal replacements, confectionery, chocolate, gel, ice cream, cereal, fruit puree, and / or solid foods, including chocolate bar, energy bar, snack bar, food bar, sauce, dip. The composition can also be mixed into solid food, for example, by sprinkling on food or mixing into it; or can be an additive that can be mixed into beverages, such as water, juice, or milk, and can contain flavors.As used herein, a smoothie is a drink made from pureed fresh fruit and / or vegetables, typically using a blender.A smoothie typically comprises a liquid base such as water, fruit juice, milk, yogurt, ice cream, or plant- and / or animal-based milk products, such as cottage cheese.A smoothie can also comprise additional ingredients, such as crushed ice, sweeteners (e.g., natural sweeteners, such as agave syrup, maple syrup, honey, or sugar, or artificial sweeteners), vinegar, flour, chocolate, or protein supplements, such as nutritional supplements.

[0070] The microorganisms in the composition should be viable, e.g., either live or in a form that supports viability, e.g., a dehydrated form that allows the yeast to be viable when rehydrated, see, e.g., U.S. Pat. No. 3,843,800; U.S. Pat. No. 3,993,783; U.S. Pat. No. 4,217,420; U.S. Pat. No. 4,341,871; U.S. Pat. No. 4,764,472; EP 0 616 030; Prepared as described in U.S. Patent No. 6,033,887; U.S. Patent No. 6,372,481; U.S. Patent Application Publication No. 20050106287; U.S. Patent Application Publication No. 20050129808; U.S. Patent Application Publication No. 20100092611; WO 2009130219; JP 2010536360; RU 2444566; and CN 102803468. See also WO 2007039586. [Example]

[0071] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0072] material and method The following materials and methods were used in the examples below. Reporter yeast strains. All genome modifications of the initial yeast strains were performed using homologous recombination of the selectable marker and transformed with at least 1 μg of linear insert DNA using standard lithium acetate transformation methods. The parent strains were either CB008 (98) for constitutive overexpression of a fluorescent reporter gene or BS004 (99) for the P2Y2-mCherry or P2Y2-apyrase gene circuits (see Table 1 for detailed strain genotypes). pFUS1-mCherry was integrated into the MFA2 locus using the plasmid pJW609, which contains the KanR marker. pFUS1 was defined as the 1636 bp immediately upstream of the Fus1 start codon. The mCherry sequence used was from Keppler-Ross, Noffz, and Dean (100), and approximately 1 kb of homologous region was used. Ste2 and Sst2 were targeted for deletion using the Trp1 and HygB selection markers, respectively, which have 180-bp flanking homology regions identical to the sequences flanking their ORFs. Plasmid pBS600, containing the selection marker LEU2 and an 800-bp homology region, replaced the five C-terminal amino acids of Gpa1 (KIGII) with a Gpa1-Gα chimera containing the C-terminal amino acids from the indicated human Gα protein. The C. albicans Adh terminator was used for pFUS1-mCherry and Gpa1-Gα gene knock-in. To generate a strain constitutively expressing mCherry, the integrative plasmid pJW609 was modified to replace the KanMX marker with HIS3 from C. glabrata, and pTDH3-mCherry was inserted into the PspOMI / BamHI sites. The linearized HIS3-pTDH3 mCherry cassette was transformed into strain CB008, and integration was selected by plating on SC-HIS. To generate a strain containing the KanMX selectable marker and constitutively expressing GFP, an integration plasmid was constructed using the MoClo yeast toolkit (101). The resulting plasmid, pBS211, contained the HO locus homology region downstream of pTDH3, the KanMX marker, and the yeast codon-optimized sfGFP gene (102).The linearized KanMX-pTDH3 sfGFP cassette was transformed into the strains in Table 1, and integration was selected by plating on YPD-G418 sulfate (200 μg / mL). All strains were confirmed by PCR and flow cytometry.

[0073] Microscopy. Yeast strain BS016 expressing the yeast codon-optimized sequence of the endogenous yeast GPCR Ste2 or human P2Y2 (obtained from ATUM) C-terminally tagged with GFP was grown to logarithmic phase in SD-URA medium. The centromeric plasmid pRS316 containing the endogenous Ste2 promoter for Ste2 expression or pTDH3 for P2Y2 expression was used, with the GFP sequence from (103). A restriction enzyme site introduced an amino acid linker (GGERGS) between the last GPCR residue and the first GFP residue. Cells were treated with concanavalin A (Sigma-Aldrich) and then plated on glass-bottom dishes (Greiner Bio-One) covered with 1 mL SD-URA medium. Cells were imaged using a Leica TCS SP8 confocal microscope.

[0074] Flow cytometry assessment of responses to ATP and UTP. Yeast strain BS016, transformed with the human P2Y2 gene in the pRS316 pTDH3 vector, was grown overnight in SC-URA liquid medium. The same strain transformed with a plasmid not containing the P2Y2 sequence (Vector) served as a negative control. Cells were cultured at OD in 600 μL SC-URA containing ATP (0–25.6 mM; pH 7.0, BioBasic) or UTP (0–3.2 mM; pH 7.0, Sigma-Aldrich). 600The antibody was diluted to 0.05 and incubated at 30°C for 6 hours. Cells were then treated with cycloheximide at a final concentration of 10 μg / mL. The mCherry signal of at least 10,000 cells was measured for each sample using a Miltenyi Biotec MACSQuant VYB. Mean mCherry fluorescence was determined using FlowJo. For dose-response assays, data were fitted to a "log(agonist) vs. response - complex slope (four parameters)" model in Prism (GraphPad). After subtracting the mCherry fluorescence signal of the vector control, fluorescence values were normalized to the wild-type P2Y2 control used in the same experiment to allow comparison between experiments performed on different days.

[0075] Directed evolution of the human P2Y2 receptor. Error-prone PCR mutagenesis was performed using the Agilent GeneMorph II random mutagenesis kit with yeast-codon-optimized human P2Y2 as the template, using a previously described method (104). The desired mutation rate of approximately three mutations per P2Y2 gene was achieved using 150 ng of template DNA and 30 cycles, and 12 randomly selected plasmids were sequenced (Table 2). Random mutants were inserted into pRS316 pTDH3 using AarI-based cloning and transformed into NEB 5-α competent Escherichia coli (E. coli) cells (New England Biolabs), resulting in >15,000 individual colonies. Cells were scraped from agar plates, mixed together, and plasmid DNA was extracted (QIAQuick Spin Miniprep Kit, Qiagen) to generate the final plasmid library. The library was transformed into yeast strain BS016 using a highly efficient lithium acetate-based method (105), generating a total library size of at least 10 times that number of colonies for multiple rounds of screening of each mutant. Transformants were incubated overnight and then transferred to OD 2000 in fresh 100 mL SC-URA liquid medium containing 100 μM ATP (pH 7.0, Biobasic). 600The solution was diluted to 0.05 and incubated at 30°C for either 18 or 6 hours (Figure 8). 6 ~10 7 Cells were gated by side and forward scatter and sorted for the highest mCherry signal (approximately 1%) using a BD Influx cell sorter (106). A total of 174 yeast colonies recovered from various sorting experiments were individually screened for their response to 100 μM UTP, 100 μM ATP, or no ligand after 6 h of incubation. Plasmids were isolated from selected colonies by first incubating with zymolase (BioShop Canada) and then extracting the plasmid DNA (QIAQuick Spin Miniprep Kit, Qiagen). Plasmid DNA was amplified using NEB 5-α competent E. coli cells (New England Biolabs), and the plasmid DNA was sequenced and transformed into fresh BS016 yeast cells for dose-response experiments.

[0076] Homology modeling of P2Y2. Modeling was performed as described by Rafehi, Neumann, Baqi, Malik, Wiese, Namasivayam, and Muller (107) using the crystal structure of the human P2Y1 receptor bound to the nucleotide antagonist MRS2500 (4XNW.pdb) as a template. The sequences of human P2Y1 and P2Y2 were aligned using Clustal Omega. Because only residues S38–F331 of P2Y1 were visible in the crystal structure, these were used as templates to generate 500 models of the corresponding P2Y2 residues L20–L313. Standard MODELLER 9.18 settings were used to retain MRS2500 in the model (108). The generated models were first analyzed based on DOPE and GA341 scores, and the top five models were manually inspected to ensure that the native disulfide bonds (C25-C278, C106-C183) were maintained. The models were then evaluated using ProSA-WEB (109) and Ramachandran plotting (110), and the final model was selected. ATP was docked into the wild-type P2Y2 homology model using the Galaxy7™ web server (111), resulting in 10 docked models. The lowest-energy model, in which the adenine ring of ATP was oriented toward the critical Y114 and F261 residues, was selected (107). Print-quality images were generated using PyMOL (Schrodinger, Inc.).

[0077] CRISPR integration of P2Y2 mutants. The pCAS plasmid expressing Cas9 and a yeast-optimized guide RNA (gRNA) was obtained from AddGene (112). The gRNA sequence was replaced with an AarI-based multiple cloning site to generate the pCAS AarI plasmid (Figure 11). This allows the use of the type IIS restriction enzyme AarI to insert any gRNA sequence (20 bp) without modifying the required nuclear localization signal or the 3' tail of the gRNA. The gRNA sequence was designed using CRISPR MultiTargeter (113) and the Off-Spotter web server (114).

[0078] [Table 2]

[0079] The forward oligo was ordered with a CTTT 5' overhang and the reverse oligo with an AAAC 5' overhang to facilitate ligation into the plasmid pCAS AarI after digestion with the AarI enzyme.

[0080] Gene cassettes containing P2Y2 variants engineered downstream of the pTDH3 promoter were assembled into the pBS600 plasmid, flanked by 800-bp homology arms for the SST2 locus. The cassettes were amplified by PCR and transformed into strain BS021 along with the pCAS AarI HygB 1143 plasmid as described by Ryan, Skerker, Maurer, Li, Tsai, Poddar, Lee, DeLoache, Dueber, Arkin, and Cate (112). Colonies were screened for mCherry expression in response to ATP, and P2Y2 integration was confirmed by sequencing.

[0081] Apyrase Genome Mining. Apyrase isolated from potato species (S. tuberosum) (RROP1; GenBank accession U58597.1) has the highest reported ATPase activity (115). The BlastPhyMe tool was used to mine the genome for homologous genes using RROP1 as the initial input sequence (116). Apyrase from wild einkorn wheat (Triticum urartu) (designated "TUAP1" in our study; GenBank accession KD039156.1) was selected because it conserves domains known to be required for apyrase function (115) and based on previous reports of wheat apyrase activity (117). Yeast codon-optimized RROP1 and TUAP1 were modified to contain an N-terminal α-factor signal peptide (the first 85 amino acids of the yeast MFα1 gene, lacking the Ste13 cleavage site) and a C-terminal HA tag (gene synthesis by ATUM).

[0082] Integration of the apyrase gene into the genome of strain P2Y2. A gene cassette containing one of the apyrase genes downstream of the pFUS1 promoter was assembled into the plasmid pBS600. The cassette was amplified by PCR and transformed into a strain containing previously integrated P2Y2, along with the plasmid pCAS AarI mCherry g664, as outlined in Ryan, Skerker, Maurer, Li, Tsai, Poddar, Lee, DeLoache, Dueber, Arkin, and Cate (112). Because mCherry had already been inserted into the MFA2 locus with pFUS1 and the C. albicans Adh terminator, the promoter and terminator of the cassette served as homologous arms. Colonies were screened for mCherry expression in response to ATP, and apyrase integration was confirmed by sequencing colonies that did not express mCherry. A second group of gene cassettes, carrying one of the apyrase genes downstream of the pTDH3 promoter, was assembled using plasmid pBS603 (pBS600 containing the HIS3 selectable marker) and flanked by 1 kb homology arms for the MFA2 locus. The cassettes were amplified by PCR and transformed into strain CB008 before plating on selective medium to generate strains BS029 (pTDH3 RROP1) and BS030 (pTDH3 TUAP1).

[0083] Western blot. Overnight cultures were cultured at OD in 50 mL YPD. 600The solution was diluted to 0.05. ATP was added to 400 μM to induce apyrase expression in the initial medium, and again at 6 and 22 hours (a final concentration of 1200 μM, assuming ATP was not degraded). All cultures were incubated at 30°C for 24 hours with shaking (225 rpm). Lysed cell samples were resolved on a 10% SDS-PAGE gel (Bio-Rad) and transferred to a PVDF membrane using a Bio-Rad Trans-Blot Turbo. The membrane was blocked overnight with Odyssey® Blocking Buffer (TBS) (LI-COR Biosciences). The following primary antibodies were used: rabbit anti-HA tag (C29F4, Cell Signaling Technology), mouse anti-PGK (459250, Invitrogen). After washing, the following secondary antibodies were used: IRDye® 680LT goat anti-mouse IgG (926-68020, LI-COR Biosciences), IRDye® 800CW goat anti-rabbit IgG (926-32211, LI-COR Biosciences). Bands were visualized using a Licor Odyssey CLx infrared imaging system (LI-COR Biosciences).

[0084] Induction of apyrase secretion with ATP. Yeast strains containing the P2Y2 mutant gene and pFUS1, which controls expression of the RROP1 apyrase, were incubated overnight in YPD medium. The cells were cultured at OD in 2 mL of fresh YPD. 600 Dilute to 0.05 and add 0-500 μM ATP (pH 7.0). 600 After 16 hours of incubation at 30°C with shaking (225 rpm), 500 μL samples were pipetted into 1.5 mL tubes and centrifuged at 2000 × g for 5 minutes to pellet the cells. Culture supernatants were then assayed for ATPase activity.

[0085] Quantification of secreted ATPase activity. The amount of ATP remaining after incubation with apyrase was determined by KinaseGlo Plus luminescence as previously described (118). ATP was added at the beginning of the culture in white 96-well microplates (#655075, Greiner Bio-One). OD 600 Five microliters of crude supernatant from the yeast culture in step 3.5 was mixed with 50 μM ATP (pH 7.0) in assay buffer (60 mM HEPES pH 6.0, 2 mM MgCl, 2 mM CaCl, 1 mM dithiothreitol, 0.1 mg / mL bovine serum albumin, 0.1 mM EDTA, and 0.01% Tween-20) in a final volume of 50 μL. The reaction was incubated at 30°C for 30 minutes and quenched by the addition of 50 μL KinaseGlo Plus (Promega). Luminescence was measured with a Fluoroskan Ascent FL microplate reader (Thermo Fisher Scientific). ATPase activity was compared to that of commercial potato apyrase (A6410, Sigma-Aldrich) incubated with ATP under the same conditions. The "percent ATP degraded" was calculated by comparison with 50 μM ATP incubated in YPD medium and assay buffer under the same conditions.

[0086] Yeast cultures for in vivo testing. Yeast strains were grown in 550 mL or 1 L YPD medium (BioShop Canada) at 30°C with shaking (225 rpm). When strains containing the KanMX resistance marker were grown, 200 μg / mL G418 sulfate antibiotic (BioShop Canada) was added to the medium. After 24 h, the cultures were centrifuged and the yeast were resuspended in fresh YPD at an OD of 92. 600 or approximately 2 × 10 9 The yeast was resuspended to 1000 cfu / mL and colony density was confirmed by plating. The yeast was stored in 800 μL aliquots at -80°C for up to 1 year.

[0087] Mice. Eight to ten week-old C57BL / 6J female (for the DSS model) or male (for the TNBS model) mice were used throughout this study. Mice were obtained from the Jackson Laboratory. All experiments were performed in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) at Brigham and Women's Hospital and Harvard Medical School.

[0088] Dextran sulfate sodium (DSS)-induced murine colitis model. IBD was induced by adding 4% dextran sulfate sodium salt to drinking water (DSS colitis grade; MP Biomedicals). Treatment was administered for 7 days, with two cycles, each with a treatment-free week in between. After the second cycle of DSS, DSS was removed and the mice were sacrificed. Animal body weight was assessed daily throughout the study.

[0089] Trinitrobenzenesulfonic acid (TNBS)-induced mouse colitis model. To induce TNBS colitis in C57BL / 6J mice, males were presensitized 1 week prior to colitis induction by applying 150 μL of presensitization TNBS solution (64% acetone (#179124, Sigma Aldrich), 16% olive oil (Sigma Aldrich #O1514), 20% 50 mg / mL TNBS (5% picrylsulfonic acid solution, Sigma Aldrich #P2297)) to their unshaven backs. After 1 week, the presensitized mice were fasted for 4 hours and then administered 100 μL of TNBS induction solution (50% ethanol, 50% 50 mg / mL TNBS) rectally. A control group was treated with 50% ethanol only. Mouse body weight was monitored daily from colitis induction at the peak of the disease until the day of euthanasia 72 hours later.

[0090] Treatment of mice with yeast: 2 × 10 8DSS mice were given 2 × 10 cfu of the corresponding yeast strain by oral gavage from day 0, which means for the entire duration of the experiment, and TNBS mice from the day of presensitization. For yeast cultures from fecal studies, mice were given 2 × 10 cfu of the corresponding yeast strain by oral gavage once. For mCherry and ATP measurement studies, mice were given 2 × 10 cfu of the corresponding yeast strain by oral gavage for 3 days prior to the study. 8 cfu of the corresponding yeast were administered by oral gavage.

[0091] Yeast culture from mouse feces: CB008, BS029, and APTM-3 yeast strains expressing a resistance gene to the antibiotic G418 were administered by oral gavage as described above. Feces were collected 2, 4, and 6 hours after oral gavage, weighed, homogenized in PBS, and cultured at 30°C in YPD agar (Cat. No. #Y1500 - Sigma Aldrich) containing 500 μg / mL G418 (Cat. No. #A1720 - Sigma Aldrich). Colony-forming units (CFU) were quantified after 72 hours.

[0092] ATP measurement in fecal contents: To evaluate the amount of ATP in fecal contents, feces from the duodenum, jejunum, ileum, cecum, and large intestine of TNBS mice treated with the corresponding yeast strain were collected 72 hours after TNBS induction and 2 hours after the last oral gavage of yeast. The fecal contents of the corresponding sections of the intestine were homogenized in PBS, and ATP measurements were performed using an ATP determination kit (#A22066, Molecular Probes) according to the manufacturer's instructions. Data were normalized to the weight of the fecal contents and the control sample.

[0093] In vivo detection of mCherry reporter yeast strains To confirm the response of our engineered P2Y2 mutants to ATP in vivo, reporter yeast expressing mCherry and constitutive GFP under the control of the most efficient P2Y2 mutant (see above) was administered to TNBS-colitis mice as described above at the peak time of disease, when more ATP is expected to be present in the intestine. The contents from specific intestinal segments were collected 2 hours after oral gavage, homogenized in YPD medium (#Y1375, Sigma-Aldrich), and cultured overnight. GFP and mCherry expression were measured by flow cytometry on a Fortessa flow cytometer (BD Biosciences), and data analysis was performed using FlowJo 10.6.1 software.

[0094] 16S Microbiome Sequencing and Analysis: Fecal samples were collected from control and TNBS-colitis mice from each yeast treatment at the end of the study. DNA was extracted using the DNeasy PowerLyzer PowerSoil Kit (#12855, Qiagen) according to the manufacturer's instructions. The 16S rRNA gene V4 region was amplified and barcoded by PCR using HotMaster Taq DNA polymerase and HotMastermix (#10847-708, VWR) and a primer library containing dual-index barcodes to allow pooling of adapters and PCR products for MiSeq sequencing. DNA was then quantified using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (#P11496, Thermo Scientific), and 100 ng of each sample was pooled and cleaned up using a QIAquick PCR Purification Kit (#28104, Qiagen). After cleanup, DNA was requantified using a Qubit fluorometric quantification kit (Thermo Scientific) and subjected to paired-end sequencing of 151-base-pair reads on an Illumina MiSeq instrument at the Harvard Medical School Biopolymer Facility, as described (119). Quality sequences were filtered by truncating reads below q20 and discarding reads shorter than 75% of their initial length, following standard protocols (120) (see Laurie for details: quality filtering and downstream analysis for α- and β-diversity, as well as compositional analysis, were performed using Quantitative Insights for Microbial Ecology Software 2 (QIIME2). Operational Taxonomic Units (OTUs) were obtained and assigned taxonomy. Distances between samples (β-diversity) were calculated using the phylogenetic-based distance UniFrac (121). Statistical testing for differential clustering of samples in PCoA plots was performed using a Permanova test with 999 permutations.Significant differences in taxa conditioned by control or active yeast treatments were determined by linear discriminant analysis effect size (LEfSe) ( 122 ).

[0095] Cytokine quantification by ELISA. Two cm of distal colon was extracted, thoroughly washed, and cultured in RPMI supplemented with 10% FBS, 100 IU / ml penicillin, 100 μg / ml streptomycin, 100 μg / ml ampicillin, and 50 μg / ml kanamycin. Supernatants were collected for later ELISA analysis. ELISA was performed according to the manufacturer's instructions (eBioscience).

[0096] Histological assessment of colitis. Colon tissues were removed and fixed in Bouin's solution (Sigma-Aldrich) and evaluated for histological assessment in a blinded fashion. Paraffin-embedded tissues were sectioned, stained with hematoxylin and eosin, and examined for evidence of colitis. A histological score (range: 0–6) was calculated based on the presence of lymphomononuclear cell infiltrates (0: absence of inflammatory foci; 1: mild presence of inflammatory foci in the mucosa; 2: presence of multiple inflammatory foci in the mucosa and submucosa; 3: evidence of transmural invasion) and intestinal architecture disruption (0: normal architecture; 1: presence of focal erosions; 2: erosions and focal ulcers; 3: extensive ulcers, tissue granulation, and / or pseudopolyps), as previously described (Erben et al. int J Clin Exp Pathol 2014).

[0097] Flow cytometry staining and acquisition. Cell suspensions were prepared from mesenteric lymph nodes. Antibodies for flow cytometry were purchased from eBioscience or BD Pharmingen and used at a concentration of 1:200 unless otherwise recommended by the manufacturer. Cells were then analyzed using a Fortessa flow cytometer (BD Biosciences and Miltenyi Biotec, respectively). Treg cells were defined as CD3+CD4+IFN-γ-IL-17-IL-10-FOXP3+.

[0098] RNA extraction and qPCR. 20 mg of distal colon was snap-frozen and then disrupted in Trizol (Invitrogen). RNA was extracted according to the manufacturer's instructions for the miRNAeasy kit (Qiagen). If required, the mRNA was further purified using an Oligotex kit (Qiagen) to remove DSS from the RNA. cDNA was prepared using a high performance RT kit (Applied Biosystems) and used for qPCR. Results were normalized to Gapdh. All primers and probes were purchased from Applied Biosystems. Gapdh Mm99999915_gl, Il17a Mm00439618_m1, Ifng Mm00801778_m1, Foxp3 Mm00475162_m1, Ccl2 Mm00441242_m1, Nos2 Mm00440502_m1, Il1b Mm00434228_m1.

[0099] Gene expression analysis using Nanostring. 100 ng of total RNA from colon tissue was analyzed using the nCounter Mouse Immunology Panel Expression Codeset according to the manufacturer's instructions (NanoString Technologies). Data were analyzed using nSolver analysis software and plotted with Heatmapper (123). Functional pathway enrichment analysis was performed using Enrichr. The combined score was calculated as c = ln(p) * z, where p is the p-value calculated using Fisher's exact test, and z is the z-score calculated using a modification of Fisher's exact test, which calculates the rank score or z-score for deviation from the expected rank (124).

[0100] Gene expression analysis by RNA sequencing: 5 ng of total RNA from colon tissue was sent for SMARTseq sequencing by Broad Technology Labs and the Broad Genomics Platform. The processed RNA-Seq data was filtered to remove genes with low read counts. Read counts were normalized using TMM normalization, and CPM (counts per million) was calculated to generate a matrix of normalized expression values. Fastq files from each RNA-Seq data sample were aligned to the Mus musculus GRCm38 transcriptome using Kallisto (v0.46.1), and the alignment results were quantified using the same software. Differential expression analysis was performed using DESeq2, and log2 fold changes were adjusted using apeGLM for downstream analysis. The Benjamini-Hochberg method was used to correct for multiple hypothesis testing. GSEA analysis was performed using the adjusted differential expression analysis results using apeGLM. Differentially expressed genes with an adjusted p-value <0.05 were analyzed with the Ingenuity® Pathway Analysis (IPA) tool to determine significantly regulated pathways.

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[0107] [Example 1] Directed evolution of the human P2Y2 receptor The P2Y2 receptor is a G protein-coupled receptor (GPCR) that senses eATP and also extracellular uridine triphosphate (eUTP) (29). First, the human P2Y2 receptor was engineered to increase its sensitivity to eATP when expressed in yeast. To establish a platform amenable to directed evolution, the human P2Y2 receptor was engineered to interact with the yeast mating pathway and the chimeric yeast Gpa1-human Gα i3 Pathway activation was monitored using a fluorescent mCherry reporter coupled via a protein and regulated by the mating-responsive FUS1 promoter (pFUS1) (Figure 1A, B, Table 1, and Figure 7A-B). Gpa1-Gα was transformed with a plasmid constitutively expressing human P2Y2. i3 The pFUS1-mCherry strain exhibited a dose-dependent response to its agonists eATP and eUTP, with logEC50 values of 3.27 μM and 2.09 μM, respectively (Fig. 1C).

[0108] Physiological eATP levels associated with inflammation have been detected in the 100 μM to high mM range (35). However, yeast expressing wild-type (WT) P2Y2 exhibit a weak response to 100 μM eATP, as determined by analysis of mCherry expression by flow cytometry (Figure 1C). Therefore, directed evolution was applied to generate yeast-expressed human P2Y2 receptor mutants that exhibit increased sensitivity to eATP. To achieve this goal, we first generated a plasmid library of human P2Y2 receptor mutants using error-prone PCR (Table 2), and then isolated yeast expressing the highest (top 1%) pFUS1-driven mCherry fluorescence after treatment with 100 μM eATP by fluorescence-activated cell sorting (FACS) (Figure 1D).

[0109] Multiple iterative rounds of FACS-based selection (36) were performed to isolate mutants exhibiting the desired increase in eATP sensitivity (Figure 8). Finally, in a post-sorting screening step, the functionality of the engineered human P2Y2 receptor mutants was further evaluated by treating selected yeast colonies with 100 μM eUTP, 100 μM eATP, or vehicle (Figure 2A). Of the 174 yeast colonies selected after multiple rounds of FACS selection, 128 colonies exhibited a stronger response to eATP than the response detected with the WT human P2Y2 receptor; 163 colonies exhibited a stronger response to eUTP. For most (but not all) of the analyzed colonies, the increased response to eATP was concomitant with the increased response to eUTP.

[0110] We focused on human P2Y2 receptor mutants that showed enhanced responses to eATP and high eATP / eUTP response ratios without constitutive expression of mCherry. Sequencing of these human P2Y2 receptor mutants revealed various genotypes with up to three nonsynonymous mutations (Table 3). Eight of the 19 human P2Y2 mutants had mutations at site F58, as defined by the Ballesteros-Weinstein transformation (37), where the first mutation is a transmembrane helix followed by a position conserved across family A GPCRs. 1.57 The mutation was also present at the nearby residue L59. 1.58 and C60 1.59 and Q165 4.57 and F307 7.54 Mutations were detected.

[0111] Ten P2Y2 mutants were selected for detailed characterization, and each mutant was named with a unique identifier based on the position of the mutated residue (Table 4). In dose-response studies, the engineered P2Y2 receptor was more responsive to both eATP and eUTP (Figure 2B). When compared to the WT human P2Y2 receptor, the selected mutants exhibited 10- to 1000-fold reductions in eATP EC50 and up to 1.8-fold reductions in the maximum conjugation pathway response. This increased sensitivity was also due to the Q165H 4.57 Except for the strains with the mutation (TM-1, TM-4), in which the maximum mating pathway response was 1.4- to 2-fold greater for eATP than for eUTP, the mutation was also detected in response to eUTP stimulation. Thus, directed evolution resulted in the generation of human P2Y2 receptor mutants with increased responsiveness to eATP.

[0112] [Table 3]

[0113] [Table 4] "Library 5" was selected for yeast transformation and FACS based on a desired mutation rate of approximately three mutations per P2Y2 sequence (approximately 2.9 mutations / kb).

[0114] [Table 5-1]

[0115] [Table 5-2]

[0116] Only unique mutants that consistently improved the WT response / sensitivity to ATP were selected for detailed characterization.

[0117] [Table 6] Maximum response values were normalized to the maximum conjugation pathway activation conferred by WT human P2Y2 receptors incubated with eATP. The dynamic range was the ratio of the highest fluorescence obtained in the presence of the indicated ligand to 10% signal saturation. The linear range was the range of ligand concentrations at which a change in signal could be detected. The minimum limit of the linear range was estimated as the ligand concentration corresponding to 10% signal saturation. Data represent the average of six colonies for eATP and three colonies for eUTP.

[0118] [Example 2] Characterization of a human P2Y2 receptor mutant with increased sensitivity to eATP Although key residues involved in nucleotide binding and activation of the human P2Y2 receptor have been identified (38, 39), the mutations detected in the 10 human P2Y2 receptor mutants we analyzed did not involve these previously identified key residues. Instead, the novel human P2Y2 receptor mutants we identified involved residues surrounding the ligand-binding pocket (A76 2.47 , N116 3.35, C119 3.38 , L162 4.54 , Q165 4.57 ), or residues located on the intracellular-facing side of the receptor (F58 1.57 , L59 1.58 , C60 1.59 , A229 ICL3 , K240 6.31 , F307 7.54 , G310 C末端 ) (Figure 3A).

[0119] To determine the molecular mechanism responsible for the increased sensitivity of selected human P2Y2 receptor mutants generated by directed evolution, we first analyzed their expression levels by microscopy and flow cytometry using C-terminally GFP-tagged receptor mutants. Human P2Y2 receptor expression in yeast was confirmed using the F58 1.57 was mutated to a smaller hydrophobic residue (C / I / L, "H1" mutant), and also in the TM-1 and TM-2 mutants (Figure 3B, C, and Table 3). Mutations in human GPCR sequences have previously been shown to improve expression in yeast (40), but these previously described mutations did not involve residues homologous to those we identified in the human P2Y2 receptor mutants generated by directed evolution. The improvement in GPCR expression was due to the P2Y2 C119S reported in our study. 3.38 Mutation similar to S90A in human adenosine A2A receptor 3.38 Mutations in transmembrane helix 1 of other GPCRs also increase stability (42), but these mutations are found in membrane-bound residues, such as F58. 1.57 , L59 1.58 , C60 1.59 Thus, our findings identify a novel role for the intracellular-facing residues of transmembrane helix 1 in the regulation of human P2Y2 expression and potentially stability.

[0120] The present inventors have identified N116S 3.35and F307S 7.54 We detected increased responsiveness and signaling in the absence of agonist (constitutive activity) in P2Y2 mutants (TM-3, H7-1, and H7-2 mutants) (Figures 2B and 3D). Interestingly, these mutants showed expression levels similar to those of the WT P2Y2 receptor. N in other family A GPCRs 3.35 Mutation at this residue has been reported to disrupt the hydrogen bond network with TM2 and TM7 residues and confer constitutive activity (43). In the engineered human P2Y2 receptor described herein, the N116S mutation was located at D79 2.50 and N298 7.45 likely disrupts a similar network with ATP, and the lack of stabilization of these inter-helical interactions results in increased signaling in the absence of agonist.

[0121] F 7.54 The residues are located immediately after the highly conserved D / NPxxY (SEQ ID NO: 18) motif required for G protein activation (44). Indeed, the F 7.54 Mutations at the conserved F in helix 8 result in constitutive activity (45). 8.50 It lacks a residue that is Y in other GPCRs. 7.53 In the human P2Y2 receptor, in addition to the conserved contact with helix 8 in the inactive state, F307 7.54 But instead this Y 7.53 (47) In summary, our findings suggest that F307S 7.54 The mutation, Y 7.53 These results suggest that the ATP-dependent ...

[0122] To further explore the mechanisms underlying the differential activity of the human P2Y2 receptor mutants, we assessed the effect of each mutation, alone or in combination with other mutations, on P2Y2 receptor activation by eATP. While certain mutations (C60Y, K240N, G310A) did not cooperate to further increase P2Y2 receptor responsiveness to eATP, others, when combined, showed deleterious effects (A76T / A229V, L162I, S359P) or positive epistasis (N116S, L59I / C119S with F58I or F307S) (Figure 3D-F). Synonymous mutations in the TM-1 mutant sequence increased P2Y2 receptor sensitivity to eATP, which could be further increased by incorporating the F58I mutation (Figure 3G). In a human P2Y2 receptor homology model docked with ATP, Q165 4.57 The Q165H mutation points toward the adenine ring of ATP. These findings suggest that the Q165H mutation aids in the receptor's interaction with ATP, resulting in increased downstream signaling. These effects of the Q165H mutation are further amplified by increased expression of the human P2Y2 receptor driven by F58I or synonymous mutations.

[0123] In summary, none of the tested mutation combinations outperformed the initial set of 10 selected human P2Y2 receptor mutants, conferring a range of improvements in sensitivity to physiological concentrations of eATP associated with inflammation. 1.57 , N116 3.35 , F307 7.54 , and Q165 4.57 Mutations in the human P2Y2 receptor (F58) increase its sensitivity to eATP and increase receptor expression. 1.57 ), stabilization of the active receptor conformation (N116 3.35 and F307 7.54 ), and improved interaction with ATP (Q165 4.57 ) contributed most highly to the ATP-dependent mechanism. 1.57All 20 amino acids were tested, revealing a diversity of eATP-induced signaling phenotypes (Figure 3H). Collectively, these findings shed new light on the molecular mechanisms regulating human P2Y2 receptor activity, and they illustrate the feasibility of applying directed evolution for the functional characterization of GPCRs.

[0124] [Example 3] eATP-driven dose-dependent induction of secreted ATPase activity in synthetic yeast Next, we engineered a therapeutic response element into a yeast synthetic gene circuit responsive to eATP. We focused on apyrase, which hydrolyzes pro-inflammatory eATP and converts it to immunosuppressive adenosine (26). We selected apyrase encoded by RROP1 in potato (Solanum tuberosum) (Figure 4A). This apyrase has potent ATPase activity and reduces inflammation when delivered intraperitoneally in a mouse model of IBD (48). Apyrase has also been identified in wheat (49). Therefore, we selected apyrase from wild einkorn (Triticum urartu) (herein referred to as TUAP1) based on its sequence homology with RROP1 in the apyrase conserved region (Figure 4A). To enable secretion by yeast, the endogenous apyrase N-terminal signal peptide was replaced with the MFα1 signal peptide, and a C-terminal HA tag was added to monitor expression (Figure 4B).

[0125] First, each modified apyrase gene under the control of a strong constitutive promoter was integrated into the yeast genome. Protein expression analysis detected multiple protein bands, suggesting that apyrase is partially degraded when expressed in yeast (Figure 4C). However, commercially available potato apyrase exhibits bands at 15 and 25 kDa, and apyrase expressed in yeast is glycosylated, resulting in multiple protein bands (50).

[0126] The culture supernatant from RROP1-expressing yeast (BS029) showed higher ATPase activity than the TUAP1-expressing yeast (BS030) (Figure 4D). When compared with commercially available apyrase, the culture supernatant from RROP1-expressing yeast showed a relative ATPase activity equivalent to approximately 280 pM of commercially available apyrase / µL crude supernatant, while that from TUAP1-expressing yeast showed an ATPase activity equivalent to <62.5 pM of commercially available apyrase / µL crude supernatant (Figure 9A-B). Therefore, RROP1 was selected as the therapeutic response element for the next study.

[0127] Subsequently, a CRISPR / Cas9-based approach was used to co-introduce a sensing element (P2Y2) and a response element (RROP1) into the genome of the same yeast strain. Six human P2Y2 receptor mutants were selected for integration into the yeast genome based on their low EC50, high dynamic range, and high maximal activation. The HygB selection marker was also removed from the yeast genome to ensure that the final strain retained uracil auxotrophy while not containing antibiotic resistance genes (an important consideration for the biocontainment and safety of engineered microorganisms).

[0128] In culture supernatants from yeast strains containing the P2Y2-RROP1 gene circuit, eATP induced ATPase enzyme activity in a dose-dependent manner (Figure 4E,F). Furthermore, ATPase activity was higher in yeast strains expressing human P2Y2 receptors engineered by directed evolution than in strains expressing the human WT P2Y2 receptor. For example, at 125 μM ATP, a 2.2- to 4.7-fold increase in ATPase activity was detected in yeast strains with engineered human P2Y2 receptors, whereas a 1.7- to 2.5-fold increase was detected at the maximum eATP concentration tested (Table 5).

[0129] Using a yeast strain constitutively overexpressing RROP1 (strain BS029), we estimated the theoretical maximum secreted ATPase. At 500 μM ATP, strains harboring engineered human P2Y2 receptor mutants exhibited 45%–69% of the ATPase activity detected in the BS029 constitutively secreting strain; yeast strains harboring human WT P2Y2 receptors exhibited only 27% of the ATPase activity. Collectively, these findings demonstrate that through a combination of directed evolution and genetic circuit engineering, we have generated yeast strains that secrete functional ATPase in response to physiological levels of eATP.

[0130] [Table 7] The apyrase data are measured as % ATP degraded (ATPase activity) and are expressed as fold difference relative to the AP-P4 strain.

[0131] [Example 4] eATP-responsive synthetic yeast probiotics attenuate intestinal inflammation We then evaluated the anti-inflammatory activity of engineered yeast probiotics using a mouse experimental model of IBD. Specifically, we tested an APTM-3 engineered yeast strain that expresses apyrase in an eATP-dependent manner. This yeast strain secretes low levels of apyrase when unstimulated, and its increased responsiveness to eATP is a known mechanism of action, due to the single mutation in P2Y2 (N116S 3.35 This yeast strain was chosen because it can directly bind to the P2Y2-RROP1 gene. Furthermore, when APTM-3 was stimulated with eATP, the ATPase activity detected was higher than or similar to that detected in other engineered P2Y2-RROP1 strains.

[0132] First, we assessed the survival of engineered yeast in the mouse gastrointestinal tract. To address this, we incorporated antibiotic resistance cassettes into the CB008, BS029, and APTM-3 engineered yeast strains to generate kanamycin-resistant CB008 KG, BS029 KG, and APTM-3 KG strains, which could be easily quantified in fecal cultures. Viability of engineered yeast in the mouse gut was assessed by oral gavage (2 × 10 8 Six hours after administration of CB008 KG, BS029 KG, or APTM-3 KG yeast (cfu), viable antibiotic-resistant yeast were detected in feces (Fig. 10A).

[0133] Increased local eATP levels have been linked to intestinal inflammation (24, 25) (Figure 10B). Therefore, to analyze the activation of P2Y2 signaling in engineered yeast during experimental colitis, we used the TM-3 yeast strain, in which activation of the mutant TM-3 P2Y2 receptor by eATP induces mCherry expression; as a control, we used the BS035 strain, which constitutively expresses mCherry (Figure 1C). For these experiments, we also incorporated an additional cassette driving constitutive GFP expression to detect administered yeast independently of eATP-driven mCherry expression. Concurrent with the increase in local eATP levels in TNBS mice, we detected mCherry expression in TM-3 engineered yeast in the cecum, proximal, and mid-colon (Figure 5A). Notably, mCherry expression was not induced in TM-3 engineered yeast administered to naive mice, in which eATP levels were not locally increased (Figure 10C). Conversely, mCherry expression was detected throughout the gastrointestinal tract of mice receiving the BS035 yeast strain, regardless of eATP levels (Figure 5A). Furthermore, when comparing mCherry expression under the control of WT or mutant TM-3 P2Y2 in vivo, higher mCherry expression was detected in yeast expressing mutant TM-3 P2Y2, highlighting the importance of P2Y2 in vitro evolution, which allows for the detection of eATP levels associated with intestinal inflammation (Figure 10D). Together, these data demonstrate that the engineered yeast can survive in the gastrointestinal tract and that the TM-3 P2Y2 mutant responds to eATP levels associated with intestinal inflammation.

[0134] The therapeutic efficacy of the engineered yeast was evaluated in an experimental model of TNBS-induced colitis in which C57BL / 6J mice were pre-sensitized and, 7 days later, colitis was induced by rectal injection of TNBS. The APTM-3 engineered yeast strain, in which apyrase is induced after activation of mutant TM-3 P2Y2 by eATP, was administered by oral gavage (2 × 10 8cfu) daily; the parental CB008 yeast strain and the BS029 engineered yeast strain, which constitutively expresses apyrase, served as controls. APTM-3 administration ameliorated TNBS-induced colitis, as demonstrated by weight loss, colon shortening assessment, and histological analysis of intestinal pathology (Figure 5B-E).

[0135] Analysis of colon samples by RNA-Seq detected a decrease in the expression of pro-inflammatory genes in mice treated with the apyrase-producing yeast strains BS029 and APTM-3; these effects were more pronounced in the APTM-3 group (Figure 5F). Indeed, treatment with the APTM-3 strain resulted in a concomitant decrease in the expression of pro-inflammatory cytokines IFNg and IL-17, which are associated with intestinal inflammation, as well as an upregulation of FoxP3+ Tregs in mesenteric lymph nodes, whereas treatment with BS029 did not (51, 52) (Figure 5G-H).

[0136] To further evaluate the therapeutic potential of engineered apyrase-expressing yeast, we used a model of colitis induced by two rounds of dextran sulfate sodium (DSS) administered in drinking water, 7 days apart (53). Yeast was orally administered starting on the day DSS administration began. Treatment with APTM-3, but not BS029, prevented the weight loss associated with DSS-induced colitis (Figure 5I). Furthermore, transcriptional analysis of colon samples by qPCR and Nanostring revealed that APTM-3 treatment resulted in decreased expression of genes associated with IBD and intestinal inflammation (51, 52, 54, 55) (Figure 5J, K). Together, these findings demonstrate that eATP-responsive yeast harboring a synthetic P2Y2-RROP1 gene circuitry attenuates intestinal inflammation.

[0137] [Example 6] eATP-responsive synthetic yeast probiotic limits colitis-associated fibrosis and dysbiosis Fibrosis contributes to the pathogenesis of IBD (56-58). Adenosine, produced by the metabolism of eATP, attenuates inflammation, whereas chronic activation of adenosine-driven purinergic signaling can promote fibrosis (26, 29). Thus, although yeast strains constitutively expressing apyrase exhibit anti-inflammatory effects, they may also promote additional pathogenic responses that can be avoided by using yeast strains that produce apyrase in response to local eATP levels. Indeed, we detected fibrotic lesions in the colons of mice treated with the control CB008 and also with the constitutive apyrase-expressing BS029 yeast strain. However, we detected significantly reduced fibrosis in mice treated with the eATP-inducible APTM-3 engineered yeast strain (Figure 6A,B). These findings suggest that controlled regulation of purinergic signaling is required to manage intestinal inflammation and avoid unwanted adverse side effects.

[0138] The microbiome plays an important role in gut physiology in health and disease (5). Furthermore, purinergic signaling is involved in gut microbiota-host communication (28, 30). Therefore, probiotics engineered to act in an inducible and localized manner are likely to minimize disruption to the gut microbiome. To investigate whether constitutive versus inducible apyrase production by engineered yeast strains differs in terms of its effect on the gut microbiome, 16S rRNA sequencing was performed on fecal samples. Consistent with previous reports (59), induction of colitis with TNBS reduced microbiome diversity within each sample, as indicated by analysis of the Shannon entropy index of α-diversity (Figure 6C). A similar reduction in microbiome diversity was detected when analyzing the effects of treatment with the BS029 yeast strain, which constitutively produces apyrase. However, treatment with an APTM-3 engineered yeast strain expressing inducible apyrase resulted in microbiome diversity levels similar to those detected in naive mice (Figure 6C).

[0139] We then analyzed beta diversity, which measures differences in microbiome composition between samples using an unweighted UniFrac distance metric that evaluates qualitative differences in microbial taxa while taking phylogenetic relationships into account. Principal coordinate analysis (PCoA) visualization of Permanova tests (Figure 6D) and pairwise UniFrac distances (Figure 6E) revealed significant differences between control and TNBS mice treated with the CB008 control or the constitutive apyrase-expressing BS029 yeast strain. Remarkably, beta diversity analysis revealed that TNBS mice treated with the APTM-3 engineered strain had microbiomes similar to those of mice without TNBS colitis, suggesting that the engineered yeast strain in which apyrase expression is induced by eATP reestablishes a healthy microbiome (Figure 6D-E).

[0140] Finally, we analyzed the taxonomic composition of the microbiome in the different treatment groups. Several taxa of commensal bacteria have been shown to be reduced in IBD and to attenuate intestinal inflammation (4, 5, 60, 61). For example, Clostrodium cluster XIVa, which has been linked to the induction of regulatory T cells (Tregs), is consistently depleted in people with IBD and acute colitis (62-64). We found that Lachnospiraceae, part of Clostridium cluster XIVa, was significantly reduced in TNBS mice treated with the CB008 and BS029 yeast strains but not in TNBS mice treated with the APTM-3 strain, which expresses inducible apyrase (Figure 6F-H). Furthermore, within the Lachnospiraceae family, the genus Roseburia was reduced in the CB008 and BS029 yeast strains but not in TNBS mice treated with APTM-3. Notably, Roseburia spp. have been shown to promote Treg development through a butyrate-dependent mechanism (65, 66). Collectively, these findings suggest that inducible production of apyrase by APTM-3-engineered yeast strains enables the anti-inflammatory effects of eATP depletion and adenosine production without the undesirable pathogenic side effects associated with fibrosis and microbiome dysregulation.

[0141] References

[0142] [Table 8-1]

[0143] [Table 8-2]

[0144] [Table 8-3]

[0145] [Table 8-4]

[0146] [Table 8-5]

[0147] [Table 8-6]

[0148] [Table 8-7]

[0149] [Table 8-8]

[0150] [Table 8-9]

[0151] [Table 8-10]

[0152] Other embodiments While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Various embodiments of the present invention are described below. 1. An isolated Saccharomyces cell that has been engineered to express one, two, or all three exogenous proteins selected from the following: (i) a mammalian P2Y purinoceptor 2 (P2Y2) protein, preferably human P2Y2; (ii) a mammalian Gα, preferably Gαi3 a mutant Gpa1 protein comprising at least five C-terminal residues derived from, wherein the mutant Gpa1 protein couples the P2Y2 protein to the yeast mating pathway; and (iii) An anti-inflammatory protein, optionally wherein the anti-inflammatory protein is mammalian, preferably human, and wherein the anti-inflammatory protein is expressed under the control of a promoter that is activated downstream of P2Y2 activation, optionally a mating-responsive promoter, and wherein the isolated Saccharomyces cells secrete the anti-inflammatory protein in the presence of extracellular adenosine triphosphate (eATP). 2. The isolated Saccharomyces cell of claim 1, which has been engineered to reduce or eliminate expression of one or more endogenous proteins selected from the group consisting of: (i) yeast GPCR α-factor pheromone receptor STE2; (ii) the GTPase-activating protein SST2, a negative regulator of pathway function; (iii) the cell cycle regulator cyclin-dependent protein serine / threonine kinase inhibitor protein FAR1; and (iv) Yeast Gα protein guanine nucleotide-binding protein subunit αGPA1. 3. The isolated Saccharomyces cell of claim 1 or 2, wherein the anti-inflammatory protein comprises a yeast-derived leader peptide that directs the protein to be secreted, and optionally lacks any signal or leader sequence endogenous to the anti-inflammatory protein. 4. An isolated Saccharomyces cell according to any one of 1 to 3 above, wherein the anti-inflammatory protein comprises apyrase, interleukin 10 (IL-10), IL-2, IL-27, IL-22, or IFN-β. 5. An isolated Saccharomyces cell according to any one of 1 to 4 above, wherein at least one of the P2Y2 protein, mutant Gpa1, or anti-inflammatory protein is expressed from a sequence that has been codon-optimized for expression in the Saccharomyces cell. 6. An isolated Saccharomyces cell according to any one of 1 to 5 above, wherein the P2Y2 contains one or more mutations that increase the expression of the anti-inflammatory protein. 7. The mutations are at residues around the ligand binding pocket (optionally A76 2.47 、N116 3.35 、C119 3.38 、L162 4.54 、Q165 4.57 ), and / or residues on the intracellular-facing side of said receptor (optionally F58 1.57 、L59 1.58 、C60 1.59 、A229 ICL3 、K240 6.31 、F307 7.54 、G310 C末端 7. The isolated Saccharomyces cell according to claim 6, wherein the cell is in the culture medium. 8. One or more mutations at residue F58 1.57 、N116 3.35 、F307 7.54 , and / or Q165 4.57 7. The isolated Saccharomyces cell according to claim 6, wherein the cell is in 9. The isolated Saccharomyces cell of claim 8, wherein the one or more mutations include F58C, Q165H, F307S, and / or N116S. 10. The isolated Saccharomyces cell according to claim 6, wherein the mutation comprises a mutation at N116. 11. The isolated Saccharomyces cell of claim 10, wherein the mutation comprises a mutation at N116 in combination with a mutation at F58 or F307. 12. The isolated Saccharomyces cell according to claim 11, wherein said mutation comprises the mutation N116S, optionally in combination with the mutation F58I or F307S. 13. The isolated Saccharomyces cell according to any one of 6 to 12 above, wherein the P2Y2 further comprises a mutation at L59 and / or C119. 14. The isolated Saccharomyces cell according to claim 13, wherein the further mutations include L59I and / or C119S. 15. The isolated Saccharomyces cell according to any one of 1 to 14 above, wherein the promoter activated downstream of P2Y2 activation is a mating-responsive promoter. 16. The isolated Saccharomyces cell according to claim 15, wherein the mating-responsive promoter is pFUS1 or pFIG1. 17. An isolated Saccharomyces cell according to any one of 1 to 14 above, wherein expression of the anti-inflammatory protein is driven by a synthetic transcription factor comprising a pheromone response domain and a DNA binding domain, which binds to a non-yeast DNA operator sequence upstream of the sequence encoding the anti-inflammatory protein. 18. An isolated Saccharomyces cell according to any one of 1 to 17 above, which is S. cerevisiae or S. boulardii. 19. A composition comprising an isolated Saccharomyces cell according to any one of 1 to 18 above, and optionally a physiologically acceptable carrier. 20. The composition according to claim 19, which is in a solid form for oral administration. 21. The composition according to claim 20, wherein the solid form comprises a tablet, pill, capsule, soft gelatin capsule, dragee, orally disintegrating / orally disintegrating tablet, or effervescent tablet. 22. The composition according to claim 19, which is in a liquid form for oral administration. 23. The composition according to claim 21, which is a drinking solution. 24. The composition according to claim 19, which is a nutritional composition optionally comprising liquid or solid food, feed, or drinking water. 25. A composition according to claim 24, wherein the nutritional composition is selected from beverages, optionally smoothies or fermented beverages, flavored beverages, yogurt, drinking yogurt, set yogurt, fruit and / or vegetable juices or concentrates thereof, fruit and vegetable juice powders, reconstituted fruit products, powders, malt or soy or cereal based beverages, breakfast cereals such as muesli flakes, spreads, meal replacements, confectionery, chocolates, gels, ice cream, cereal, fruit and / or chocolate bars, energy bars, snack bars, food bars, sauces, dips, and sports supplements including dairy and non-dairy based sports supplements. 26. A method for reducing inflammation in a subject, comprising administering to the subject an effective amount of an isolated Saccharomyces cell described in any one of 1 to 18 above or a composition described in any one of 19 to 25 above. 27. The method according to claim 26, wherein the subject has or is at risk of developing inflammatory bowel disease (IBD). 28. An isolated Saccharomyces cell according to any one of 1 to 18 above, or a composition according to any one of 19 to 25 above, for use in a method for reducing inflammation in a subject. 29. An isolated Saccharomyces cell or composition for use according to claim 28, wherein the subject has or is at risk of developing inflammatory bowel disease (IBD). 30. Residues around the ligand binding pocket (optionally, A76 2.47 、N116 3.35 、C119 3.38 、L162 4.54 、Q165 4.57 ), and / or residues on the intracellular-facing side of the receptor (optionally F58 1.57 、L59 1.58 、C60 1.59 、A229 ICL3 、K240 6.31 、F307 7.54 、G310 C末端 ) an engineered mammalian P2Y purinergic receptor 2 (P2Y2) protein containing one or more mutations in the 31. One or more mutations at residue F58 1.57 、N116 3.35 、F307 7.54 , and / or Q165 4.57 31. The engineered mammalian P2Y2 according to claim 30, wherein the P2Y2 is in 32. The engineered mammalian P2Y2 according to claim 31, wherein said one or more mutations comprise residues F58C, Q165H, F307S, and / or N116S. 33. The engineered mammalian P2Y2 according to claim 30, wherein said mutation comprises a mutation at N116. 34. The engineered mammalian P2Y2 according to claim 33, wherein said mutation comprises a mutation at N116 in combination with a mutation at F58 or F307. 35. The engineered mammalian P2Y2 according to claim 34, wherein said mutation comprises the mutation N116S, optionally in combination with the mutation F58I or F307S. 36. The engineered mammalian P2Y2 according to any one of claims 30 to 35, wherein said P2Y2 further comprises a mutation at L59 and / or C119. 37. The engineered mammalian P2Y2 according to claim 36, wherein said further mutations include L59I and / or C119S. 38. An isolated nucleic acid sequence encoding an engineered mammalian P2Y2 according to any one of 30 to 37 above. 39. A host cell comprising the isolated nucleic acid sequence described in 35 above, and optionally expressing an engineered mammalian P2Y2 described in any of 30 to 37 above. 40. The host cell according to claim 39, wherein the cell is a Saccharomyces cell and the isolated nucleic acid sequence is codon-optimized for expression in the Saccharomyces cell.

Claims

1. An isolated Saccharomyces cell that has been engineered to express: (i) mammalian P2Y purinoceptor 2 (P2Y2) protein; (ii) a mutant Gpa1 protein comprising at least five C-terminal residues from a mammalian Gα, wherein the mutant Gpa1 protein couples the P2Y2 protein to the yeast mating pathway; and (iii) an anti-inflammatory protein, wherein the anti-inflammatory protein is expressed under the control of a promoter that is activated downstream of P2Y2 activation, and wherein the isolated Saccharomyces cell secretes the anti-inflammatory protein in the presence of extracellular adenosine triphosphate (eATP).

2. 2. The isolated Saccharomyces cell of claim 1, wherein the P2Y2 protein is human P2Y2.

3. The mutant Gpa1 protein is a mammalian Gα ｉ３ 3. The isolated Saccharomyces cell of claim 1 or 2, comprising at least five C-terminal residues derived from said Saccharomyces cell.

4. 4. The isolated Saccharomyces cell of claim 1, wherein the anti-inflammatory protein is a mammalian anti-inflammatory protein.

5. 5. The isolated Saccharomyces cell of claim 1, wherein the anti-inflammatory protein is a human anti-inflammatory protein.

6. 6. The isolated Saccharomyces cell of any one of claims 1 to 5, which has been engineered to reduce or eliminate expression of one or more endogenous proteins selected from the group consisting of: (i) yeast GPCR α-factor pheromone receptor STE2; (ii) the GTPase-activating protein SST2, a negative regulator of pathway function; (iii) cell cycle regulator cyclin-dependent protein serine / threonine kinase inhibitory protein FAR1; and (iv) Yeast Gα protein guanine nucleotide-binding protein subunit αGPA1.

7. 7. The isolated Saccharomyces cell of claim 1, wherein the anti-inflammatory protein comprises a yeast-derived leader peptide that directs the protein to be secreted.

8. 8. The isolated Saccharomyces cell of claim 7, wherein the yeast-derived leader peptide lacks any signal or leader sequence endogenous to the anti-inflammatory protein.

9. 9. The isolated Saccharomyces cell of claim 1, wherein the anti-inflammatory protein is apyrase, interleukin 10 (IL-10), IL-2, IL-27, IL-22, or IFN-β.

10. 10. The isolated Saccharomyces cell of any one of claims 1 to 9, wherein at least one of the P2Y2 protein, mutant Gpa1, or anti-inflammatory protein is expressed from a sequence that is codon-optimized for expression in the Saccharomyces cell.

11. the P2Y2 comprises one or more mutations relative to NP_002555.4 - SEQ ID NO: 13 that increase expression of the anti-inflammatory protein, the mutations being: in residues surrounding the ligand-binding pocket, the mutations being A76T, N116S, C119S, L162I, Q165H, or a combination thereof; and / or the mutation is in a residue on the intracellular-facing side of the P2Y2, and the mutation is F58I or F58C, L59I, C60Y, A229V, K240N, F307S, G310A, or a combination thereof; 11. The isolated Saccharomyces cell of any one of claims 1 to 10.

12. The isolated Saccharomyces cell of claim 11, wherein the one or more mutations are selected from F58I or F58C, N116S, F307S, Q165H, or a combination thereof.

13. 13. The isolated Saccharomyces cell of claim 12, wherein the one or more mutations comprise F58C, Q165H, F307S, and / or N116S.

14. 12. The isolated Saccharomyces cell of claim 11, wherein the mutation comprises N116S.

15. 15. The isolated Saccharomyces cell of claim 14, wherein the mutation comprises N116S in combination with F58I, F58C, or F307S.

16. 16. The isolated Saccharomyces cell of claim 15, wherein the mutation comprises a N116S mutation in combination with an F58I or F307S mutation.

17. 17. The isolated Saccharomyces cell of any one of claims 11 to 16, wherein the P2Y2 further comprises the mutations L59I and / or C119S.

18. 18. The isolated Saccharomyces cell of claim 1, wherein the promoter that is activated downstream of P2Y2 activation is a mating-responsive promoter.

19. 19. The isolated Saccharomyces cell of claim 18, wherein the mating-responsive promoter is pFUS1 or pFIG1.

20. 18. The isolated Saccharomyces cell of any one of claims 1 to 17, wherein expression of the anti-inflammatory protein is driven by a synthetic transcription factor comprising a pheromone response domain and a DNA binding domain that binds to a non-yeast DNA operator sequence upstream of the sequence encoding the anti-inflammatory protein.

21. 21. The isolated Saccharomyces cell of any one of claims 1 to 20, which is S. cerevisiae or S. boulardii.

22. 22. A composition comprising the isolated Saccharomyces cell of any one of claims 1 to 21 and a physiologically acceptable carrier.

23. 23. The composition of claim 22, in a solid form for oral administration.

24. 24. The composition of claim 23, wherein the solid form comprises a tablet, pill, capsule, soft gelatin capsule, dragee, orodisintegrant / orodisintegrating tablet, or effervescent tablet.

25. 23. The composition of claim 22 in liquid form for oral administration.

26. 26. The composition of claim 25, which is a drinking solution.

27. 23. The composition of claim 22, which is a nutritional composition.

28. 28. The composition of claim 27, wherein the nutritional composition comprises a liquid or solid food, feed, or drinking water.

29. 29. The composition of claim 27 or 28, wherein the nutritional composition is selected from beverages, breakfast cereals, spreads, meal replacements, confectioneries, chocolates, gels, ice creams, cereals, fruit and / or chocolate bars, energy bars, snack bars, food bars, sauces, dips, and sports supplements.

30. The beverage comprises a smoothie or fermented beverage, a flavored beverage, a yogurt, a drinking yogurt, a set yogurt, a fruit and / or vegetable juice or concentrate thereof, a fruit and vegetable juice powder, a reconstituted fruit product, a powder, a malt or a soy or cereal based beverage, the breakfast cereal comprises muesli flakes, and / or Sports supplements include dairy and non-dairy based sports supplements; 30. The composition of claim 29.

31. 31. A composition comprising the isolated Saccharomyces cell of any one of claims 1 to 21, or the composition of any one of claims 22 to 30, for use in a method of reducing inflammation in a subject.

32. 32. The composition of claim 31, wherein the subject has or is at risk of developing inflammatory bowel disease (IBD).

33. NP_002555.4 - An engineered mammalian P2Y purinoceptor 2 (P2Y2) protein comprising one or more mutations relative to SEQ ID NO: 13 that increase expression of an anti-inflammatory protein, wherein the mutations are: in residues surrounding the ligand-binding pocket, the mutations being A76T, N116S, C119S, L162I, Q165H, or a combination thereof; and / or 1. An engineered mammalian P2Y2, wherein the mutation is in a residue on the intracellular-facing side of said P2Y2 and is F58I or F58C, L59I, C60Y, A229V, K240N, F307S, G310A, or a combination thereof.

34. 34. The engineered mammalian P2Y2 of claim 33, wherein the one or more mutations are selected from residues F58I or F58C, N116S, F307S, Q165H, or a combination thereof.

35. 35. The engineered mammalian P2Y2 of claim 34, wherein the one or more mutations comprise residues F58C, Q165H, F307S, and / or N116S.

36. 34. The engineered mammalian P2Y2 of claim 33, wherein the mutation comprises N116S.

37. 37. The engineered mammalian P2Y2 of claim 36, wherein the mutation comprises N116S in combination with F58I, F58C, or F307S.

38. 38. The engineered mammalian P2Y2 of claim 37, wherein the mutation comprises the mutation N116S in combination with the mutation F58I or F307S.

39. 39. The engineered mammalian P2Y2 of any one of claims 33 to 38, wherein said P2Y2 further comprises the mutations L59I and / or C119S.

40. 40. An isolated nucleic acid encoding the engineered mammalian P2Y2 of any one of claims 33 to 39.

41. 41. A host cell comprising the isolated nucleic acid of claim 40.

42. 42. A host cell according to claim 41, which expresses the engineered mammalian P2Y2 of any one of claims 33 to 39.

43. 43. The host cell of claim 41 or 42, wherein the cell is a Saccharomyces cell and the isolated nucleic acid is codon-optimized for expression in the Saccharomyces cell.

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