Engineered microbes as delivery vehicles of modulatory molecules of the enteroendocrine system

Abstract

The technology described herein is directed to probiotic microbes engineered to secrete at least one enteroendocrine modulating polypeptide, which can be a sweet protein. In some embodiments, the probiotic microbe secretes the enteroendocrine modulating polypeptide in the distal colon. Also described herein are nucleic acids or vectors comprising a coding sequence for the enteroendocrine modulating polypeptide. Also described herein are compositions comprising the engineered probiotic microbe. Methods of using such probiotic microbes and compositions are also disclosed, including methods for modulating enteroendocrine cells and methods for treating metabolic diseases.

Description

ENGINEERED MICROBES AS DELIVERY VEHICLES OF MODULATORY MOLECULES OF THE ENTEROENDOCRINE SYSTEMCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 643,668 filed May 7, 2024, the contents of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on April 28, 2025, is named 002806-000129WOPT_SL.xml and is 7,831 bytes in size.TECHNICAL FIELD

[0003] The technology described herein relates to engineered microbes as delivery vehicles of modulatory molecules of the enteroendocrine system.BACKGROUND

[0004] The prevalence of metabolic diseases (e.g., diabetes, obesity, liver disease, among others) is steadily increasing worldwide. This trend calls for new therapeutic approaches to help a broad spectrum of patients, starting at the early onset of metabolic dysregulation. The gastrointestinal (GI) tract is the master regulator of metabolic health, acting through endocrine signaling and the gut-brain axis, and therefore is considered a key system to promote metabolic health. In recent years, the gut microbiota, a collection of diverse intestinal microorganisms, has been recognized as a key driver of multiple gut-centered systemic functions, revealing a previously unappreciated role of microbial cells in the interplay between heath and disease (see e.g., Fan, Y. 2021, Nat Rev Microbiol 19, 55-71). The structural components and metabolic products derived from the gut microbiota are fundamental for the proper function of not only the digestive system, but also for eliciting systemic effects that influence the immune, endocrine, and neurologic systems. The need for antibiotic treatment and the consumption of diets high in sugars and fats can lead to changes in the diversity, composition, and activity of the gut microbiota, which results in a pathological state called dysbiosis (see e.g., Fan, Y. 2021). Preventing and / or restoringmicrobiota-dependent gut functions lost due to dysbiosis is a therapeutic target for the management of multiple human diseases. Current approaches to restore physiological functions that stem from the microbiota activity in the gut have involved the oral supplementation of single or multiple bacterial strains, which usually lack robustness in their activity and / or a defined mechanism of action. More recent approaches have involved the transference of donor-derived fecal preparations that result in greater microbiota restoration efficacy but face safety issues and supply limitations (see e.g., Fan, Y. 2021). To tackle these challenges, engineered live biotherapeutic products (eLBP) can be used that replace key microbiota functions as an approach to treat gut-centered pathologies that have not been addressable using conventional therapeutic approaches.

[0005] In the gut, specialized epithelial cells sense nutrients and microbiota-generated metabolites, and as response, they orchestrate a complex downstream response involving the secretion of potent hormones necessary for proper metabolic function. A dysbiotic gut microbiota displays impairment in functionalities that are necessary for proper enteroendocrine function, including glucose homeostasis. Obesity and metabolic changes occurring with the development of type 2 diabetes are associated with a decline in the secretion of the enteroendocrine hormones from intestinal L-cells, including GLP-1 (see e.g., Greiner TU, 2016, Mol Metab. (9):753-758). There is evidence that the activity of the gut microbiota stimulates GLP-1 production from L-cells in the large intestine; however, dysbiosis in type 2 diabetes patients precludes this endogenous stimulation, which can result in poor glucose control. Elevating GLP-1 levels has been recognized as an important therapeutic target in type 2 diabetes and pharmacologic agents, namely GLP-1 receptor agonists and DDP-IV inhibitors have been developed with success in clinical settings (see e.g., Drucker DJ, 2017, J Clin Invest. 127(12): 4217-4227). However, GLP-1 receptor agonists only exert their effects through an endocrine route and therefore they require supraphy si ologi cal concentrations that render them less efficacious and more prone to serious side effects.SUMMARY

[0006] The technology described herein addresses problems related to delivery of enteroendocrine modulators using probiotic microbes to enhance the secretion of gut hormones using endogenous pathways that result in more relevant physiological responses. The technology described herein is directed to probiotic microbes, such as a probiotic yeast,engineered to secrete at least one enteroendocrine modulating polypeptide, such as, but not limited to a sweet protein. In some embodiments, the probiotic microbe secretes the enteroendocrine modulating polypeptide in the distal colon. Also described herein are nucleic acids or vectors comprising a coding sequence for the enteroendocrine modulating polypeptide. Also described herein are compositions comprising the engineered probiotic microbe. Methods of using such probiotic microbes and compositions are also disclosed, including methods for modulating enteroendocrine cells and methods for treating metabolic diseases.

[0007] In one aspect, described herein is a probiotic microbe engineered to secrete an enteroendocrine modulating polypeptide.

[0008] In some embodiments of any of the aspects, the probiotic microbe is a yeast.

[0009] In some embodiments of any of the aspects, the yeast is selected from the group consisting of Saccharomyces cerevisiae (5. cerevisiae), Saccharomyces boulardii (S. boulardii), and Saccharomyces unisporus (S. unisporus).

[0010] In some embodiments of any of the aspects, the probiotic microbe is a bacterium.

[0011] In some embodiments of any of the aspects, the bacterium belongs to a genus selected from the group consisting of Lactobacillus, Pediococcus, Streptococcus, Bacillus, Enter occous, Escherichia, Vibrio, Bacteroides, and Bifidobacterium .

[0012] In some embodiments of any of the aspects, the enteroendocrine modulating polypeptide comprises an enteroendocrine receptor agonist.

[0013] In some embodiments of any of the aspects, the enteroendocrine modulating polypeptide comprises an enteroendocrine receptor antagonist.

[0014] In some embodiments of any of the aspects, the enteroendocrine receptor is selected from the group consisting of taste receptor type 1 member 1 (TAS1R1), taste receptor type 1 member 2 (TAS1R2), taste receptor type 1 member 3 (TAS1R3), G Protein- Coupled Receptor 93 (GPR93), G-protein-couple receptor 6A (GPCR6A), G-protein-coupled receptor 142 (GPR142), calcium-sensing receptor (CaSR), Metabotropic Glutamate Receptor 1 (mGluRl), Metabotropic Glutamate Receptor 4 (mGluR4), PEPT1, and cholecystokinin 1 receptor (CCK-1R).

[0015] In some embodiments of any of the aspects, the enteroendocrine modulating polypeptide comprises a taste receptor type 1 member 3 (TAS1 / R3) receptor agonist and / or a taste receptor type 1 member 2 (TAS1 / R2) receptor agonist.

[0016] In some embodiments of any of the aspects, the TAS1 / R3 receptor and / or TAS1 / R2 agonist comprises a sweet protein.

[0017] In some embodiments of any of the aspects, the sweet protein comprises a singlechain polypeptide version of monellin.

[0018] In some embodiments of any of the aspects, the single-chain version of monellin comprises MNEI.

[0019] In some embodiments of any of the aspects, the enteroendocrine modulating polypeptide comprises a hormone selected from the group consisting of cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), glucagon- like peptide 2 (GLP-2), glicentin, oxyntomodulin, Peptide YY (PYY), secretin, Substance P (SP), xenin, glucose-dependent insulinotropic polypeptide (GIP), neurotensin, motilin, Gcg (GLP1 precursor), ghrelin, somatostatin, gastrin, oxyntomodulin (OXM), and b-casomorphin.

[0020] In some embodiments of any of the aspects, the enteroendocrine modulating polypeptide comprises a peptide antagonist of Neuropeptide Y Receptor Y1 (NpylR) or a peptide antagonist of dipeptidyl peptidase IV (DPP -IV).

[0021] In some embodiments of any of the aspects, the nucleic acid sequence encoding the enteroendocrine modulating polypeptide is codon-optimized for expression in the host probiotic microbe.

[0022] In one aspect, described herein is a nucleic acid encoding and capable of expressing or directing the expression of an enteroendocrine modulating polypeptide, wherein the enteroendocrine modulating polypeptide is codon-optimized for expression in a yeast, and / or the enteroendocrine modulating polypeptide is operably linked to a yeast regulatory element.

[0023] In one aspect, described herein is a nucleic acid encoding and capable of expressing an enteroendocrine modulating polypeptide, wherein the enteroendocrine modulating polypeptide is codon-optimized for expression in a bacterium, and / or the enteroendocrine modulating polypeptide is operably linked to a bacterial regulatory element.

[0024] In one aspect, described herein is a vector comprising a nucleic acid as described herein.

[0025] In one aspect, described herein is a probiotic microbe comprising a nucleic acid as described herein or a vector as described herein.

[0026] In one aspect, described herein is a composition comprising a probiotic microbe as described herein, or a nucleic acid as described herein, or a vector as described herein.

[0027] In some embodiments of any of the aspects, the probiotic microbe is dried and viable.

[0028] In some embodiments of any of the aspects, the probiotic microbe secretes the enteroendocrine modulating polypeptide in the stomach, duodenumjejunum, ileum, cecum, mid-colon, and / or distal colon of a subject.

[0029] In some embodiments of any of the aspects, the probiotic microbe secretes the enteroendocrine modulating polypeptide in the distal colon of a subject.

[0030] In some embodiments of any of the aspects, the probiotic microbe is formulated for delivery to the stomach, duodenum ejunum, ileum, cecum, mid-colon, and / or distal colon of a subject.

[0031] In some embodiments of any of the aspects, the probiotic microbe is formulated for delivery to the distal colon of a subject.

[0032] In one aspect, described herein is a pharmaceutical composition comprising a probiotic microbe as described herein, or a nucleic acid as described herein, or a vector as described herein, in combination with a pharmaceutically acceptable carrier.

[0033] In some embodiments of any of the aspects, the microbe is in spore or dried viable form.

[0034] In some embodiments of any of the aspects, the probiotic microbe is encapsulated.

[0035] In some embodiments of any of the aspects, the encapsulation comprises an enteric coating.

[0036] In one aspect, described herein is a food composition comprising a probiotic microbe as described herein or a composition as described herein.

[0037] In some embodiments of any of the aspects, the food composition further comprises a prebiotic that supports growth or colonization by the probiotic microbe.

[0038] In some embodiments of any of the aspects, the prebiotic is selected from amino acid (e.g., arginine, glutarate, and ornithine), biotin, fructooligosaccharide, galactooligosaccharide, hemi cellulose (e.g., arabinoxylan, xylan, xyloglucan, glucomannan), inulin, chitin, lactulose, mannan oligosaccharide, oligofructose-enriched inulin, gum (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectin (e.g., xylogal actouronan, citrus pectin, apple pectin, and rhamnogal acturonan-I), dietary fiber (e.g., soy fiber, sugarbeet fiber, pea fiber, com bran, and oat fiber), xylooligosaccharide, and polyamine (e.g., spermidine, putrescine).

[0039] In one aspect, described herein is a medical food comprising a probiotic microbe as described herein.

[0040] In one aspect, described herein is a supplement comprising a probiotic microbe as described herein.

[0041] In one aspect, described herein is a method of enteroendocrine modulation, the method comprising administering an enteroendocrine-modulating amount of a probiotic microbe as described herein, a composition as described herein, a pharmaceutical composition as described herein, a food composition as described herein, a medical food as described herein, or a supplement as described herein to a subject in need of enteroendocrine modulation.

[0042] In some embodiments of any of the aspects, the enteroendocrine modulating polypeptide modulates metabolic energy homeostasis in the subject.

[0043] In some embodiments of any of the aspects, metabolic energy homeostasis modulation comprises modulation of glucose levels, modulation of metabolism, modulation of type 2 diabetes, weight management, satiety, gastric emptying, gut motility, nutrient absorption, gut hormone signaling, and / or management of dyslipidemia.

[0044] In some embodiments of any of the aspects, the probiotic microbe or the composition is administered daily.

[0045] In one aspect, described herein is a method of modulating blood glucose homeostasis, the method comprising administering an enteroendocrine-modulating amount of a probiotic microbe engineered to secrete a polypeptide agonist of the T1R2-T1R3 sweet receptor or to secrete a polypeptide agonist of the GLP-1 receptor.

[0046] In one aspect, described herein is a method of treating type II diabetes, the method comprising administering an enteroendocrine-modulating amount of a probiotic microbe engineered to secrete an agonist of the GLP-1 receptor.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Fig. 1 shows delivery of a luminescent reporter enzyme throughout the mouse gastrointestinal tract using an engineered S. boulardii strain. A total of 12 mice were dosed with 108CFUs of the engineered strain by oral gavage. Groups of 4 mice were sacrificed at 2- hours, 5-hours, and 23-hours post-dosing for organ and fecal sample harvesting. Cell counts were obtained using the drop count method in YTG plates incubated at 30°C for 36 hours.Luminesce signal was obtained from homogenized fecal samples using the NANOGLO™ (PROMEGA) system.

[0048] Fig. 2 shows a visualization of MNEI secretion to the culture supernatant using SDS-PAGE.

[0049] Fig. 3A-3C shows metabolic profiling of diet-induced obese (DIO) mice subject to oral treatment with ELITE-031. Fig. 3A shows the experimental design for the investigation of modulation of enteroendocrine metabolic markers in DIO and normal diet (ND) mice. DIO-ELITE-031, n=9, DIO-ELITE-029, n=10, DIO-PBS n=l l and Normal Diet- PBS n=5. Fig. 3B shows quantification of the principal metabolic markers, gut hormones and adipokines related to the mouse enteroendocrine system. Fig. 3C shows TSNE analysis of metabolic profiles identifies clusters that indicate distinct modulation of the enteroendocrine system by the different oral treatments.

[0050] Fig. 4A-4C shows glycemic response of DIO mice treated with ELITE-031. Fig. 4A shows measurement of basal glucose concentration in 6-hour fasted mice. Fig. 4B shows oral glucose tolerance test (OGTT) curves upon the gavage of a 2 mg / kg bolus of glucose. Fig. 4C shows determination of the area under the curve (AUC) of the glycemic responses to OGTT. Number of animals per group are as follows: DIO-ELITE-031, n=9, DIO-ELITE-029, n=10, DIO-PBS n=l 1 and Normal Diet (ND)-PBS n=5. Bars represent arithmetic means and standard deviations; samples are compared by ANOVA, * represents a p-value p<0.05, ** represents a p-value p<0.01, *** represents a p-value p<0.001.DETAILED DESCRIPTION

[0051] The technology described herein is directed to probiotic microbes, such as a probiotic yeast, engineered to secrete an enteroendocrine modulating polypeptide, which can be an agonist or antagonist for a receptor expressed by an enteroendocrine cell or a hormone secreted by an enteroendocrine cell. As a non-limiting example, the enteroendocrine modulating polypeptide can be a sweet protein. In some embodiments, the probiotic microbe secretes the enteroendocrine modulating polypeptide in the distal colon. Also described herein are nucleic acids or vectors comprising a coding sequence for the enteroendocrine modulating polypeptide, where such a coding sequence can be codon-optimized for the microbe and / or operably linked to a regulatory element active in the microbe. Also described herein are compositions, such as food compositions, medical foods, supplements, formulations, or pharmaceutical compositions comprising the engineered probiotic microbe.Methods of using such probiotic microbes and compositions are also disclosed, including methods for modulating enteroendocrine cells and methods for treating metabolic diseases, such as diabetes, obesity, or liver disease.Enteroendocrine Modulating Polypeptides

[0052] Described herein in multiple aspects are probiotic microbes engineered to secrete at least one enteroendocrine modulating polypeptide. In some embodiments, the probiotic microbe secretes 1, 2, 3, 4, 5, or more enteroendocrine modulating polypeptides. As used herein, the term “enteroendocrine modulating polypeptide” refers to a protein capable of increasing or decreasing at least one enteroendocrine activity of an enteroendocrine cell in the gastrointestinal system. Enteroendocrine cells are cells in the gastrointestinal tract and pancreas with endocrine function, which produce gastrointestinal hormones or peptides in response to various stimuli; non-limiting examples of such hormones include somatostatin, motilin, cholecystokinin, neurotensin, vasoactive intestinal peptide, and enteroglucagon. The enteroendocrine cells can release such hormones into the bloodstream for systemic effect, diffuse them as local messengers, or transmit them to the enteric nervous system to activate nervous responses. In some embodiments, an enteroendocrine modulating polypeptide, secreted by a probiotic microbe as described herein, can bind to at least one receptor expressed on an enteroendocrine cell and thus increase or decrease the cell’s activity, such as secretion of a hormone.

[0053] In some embodiments, the enteroendocrine cell modulated by the enteroendocrine modulating polypeptide is located in the stomach, duodenumjejunum, ileum, cecum, midcolon, and / or distal colon of a subject. In some embodiments, the enteroendocrine cell modulated by the enteroendocrine modulating polypeptide is an intestinal enteroendocrine cell. In some embodiments, the enteroendocrine cell modulated by the enteroendocrine modulating polypeptide is located in the colon, such as the distal colon. In some embodiments, the enteroendocrine cell modulated by the enteroendocrine modulating polypeptide is a K cell, an L cell, an I cell, a G cell, an enterochromaffin cell, an enterochromaffin-like cell, an N cell, an S cell, a D cell, or an M cell.

[0054] In some embodiments, the enteroendocrine modulating polypeptide comprises an enteroendocrine receptor agonist; for example, the polypeptide agonist can bind to the receptor and induce downstream signaling of the receptor. In some embodiments, the enteroendocrine modulating polypeptide comprises an enteroendocrine receptor antagonist; for example, the polypeptide antagonist can bind to the receptor and prevent or reducebinding of agonists for the receptor, thus decreasing or inhibiting downstream signaling of the receptor. In some embodiments, the enteroendocrine modulating polypeptide specifically binds to a receptor expressed on an enteroendocrine cell. Non-limiting examples of enteroendocrine receptors (i.e., receptors expressed on the cell membrane of enteroendocrine cells) include: taste receptor type 1 member 1 (TAS1R1), taste receptor type 1 member 2 (TAS1R2), taste receptor type 1 member s (TAS1R3), G Protein-Coupled Receptor 93 (GPR93), G-protein-coupled receptor 6A (GPCR6A), G-protein-coupled receptor 142 (GPR142), calcium-sensing receptor (CaSR), Metabotropic Glutamate Receptor 1 (mGluRl), Metabotropic Glutamate Receptor 4 (mGluR4), PEPT1, and cholecystokinin 1 receptor (CCK-1R).

[0055] In some embodiments, the enteroendocrine receptor and its agonist (secreted by the probiotic microbe) are selected from Table 1. In some embodiments, the enteroendocrine receptor agonist comprises a peptide (e.g., at most 40-50 amino acids long). In some embodiments, the enteroendocrine receptor and its antagonist (secreted by the probiotic microbe) are selected from Table 1. In some embodiments, the enteroendocrine receptor antagonist comprises a peptide (e.g., at most 40-50 amino acids long). In some embodiments, the probiotic microbe is engineered to secrete an enzyme that produces an agonist or antagonist listed in Table 1.

[0056] Table 1 : Exemplary enteroendocrine receptors, exemplary enteroendocrine receptor agonist(s), and exemplary enteroendocrine receptor antagonist(s).

[0057] For more information concerning enteroendocrine receptors, agonists, and antagonists, see e.g., Bai et al. “Enteroendocrine cell types that drive food reward and aversion,” eLife 2022, 11 : e74964; Hayashi et al. “Enteroendocrine cell lineages that differentially control feeding and gut motility,” eLife 2023,12: e78512; Caron et al. “Protein Digestion-Derived Peptides and the Peripheral Regulation of Food in-take,” Front Endocrinol (Lausanne), 2017, 8: 85; Liu et al. “Engineering yeast phospholipid metabolism for de novo oleoylethanolamide production,” Nature Chemical Biology Vol. 16, 2020: 197-205; Santos- Hernandez et al. “Intestinal Signaling of Proteins and Digestion-Derived Products Relevant to Satiety,” J Agric Food Chem 2018, 66(39): 10123-10131; Wooding et al. “Bitter taste receptors,” Evol Med Public Health. 2021; 9(1): 431-447; Richter et al. “Bitter Peptides YFYPEL, VAPFPEVF, and YQEPVLGPVRGPFPIIV, Released during Gastric Digestion of Casein, Stimulate Mechanisms of Gastric Acid Secretion via Bitter Taste Receptors TAS2R16 and TAS2R38,” J Agric Food Chem. 2022 Sep 21; 70(37): 11591-11602;Maehashi et al. “Sweetness of lysozymes,” Biosci Biotechnol Biochem. 1998 Mar;62(3):605- 606; the contents of each of which are incorporated herein by reference in their entireties.

[0058] In some embodiments, the enteroendocrine modulating polypeptide comprises an agonist of at least one GPCR taste receptor (see e.g., Table 1). In some embodiments, the enteroendocrine modulating polypeptide comprises an agonist of taste receptor type 1 member 3 (also referred to interchangeably as TAS1R3, TAS1 / R3, or T1R3). The TAS1 / R3 protein is a G protein-coupled receptor with seven trans-membrane domains and is a component of the heterodimeric amino acid taste receptor TAS1R1+3 and sweet taste receptor TAS1R2+3. A homo-dimer of TAS1R3 is also sensitive to natural sugar substances. In some embodiments, the enteroendocrine modulating polypeptide comprises an agonist of taste receptor type 1 member 2 (also referred to interchangeably as TAS1R2, TAS1 / R2, or T1R2). In some embodiments, the enteroendocrine modulating polypeptide comprises an agonist of TAS1R2 and TAS1R3.

[0059] In some embodiments, the TAS1 / R3 receptor comprises SEQ ID NO: 1, SEQ ID NO: 2 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to SEQ ID NO: 1 or SEQ ID NO: 2, which maintains its function (e.g., binding to an agonist, such as a sweet protein).

[0060] SEQ ID NO: 1, TAS1 / R3 receptor UNIPROT Q7RTX0, Homo sapiens, 852 amino acids (aa)MLGPAVLGLSLWALLHPGTGAPLCLSQQLRMKGDYVLGGLFPLGEAEEAGLRSRTRPSSPVCTRFSSNGLLWALAMKMAVEEINNKSDLLPGLRLGYDLFDTCSEPVVAMKPSLMFLAKAGSRDIAAYCNYTQYQPRVLAVIGPHSSELAMVTGKFFSFFLMPQVSYGASMELLSARETFPSFFRTVPSDRVQLTAAAELLQEFGWNWVAALGSDDEYGRQGLSIFSALAAARGICIAHEGLVPLPRADDSRLGKVQDVLHQVNQSSVQVVLLFASVHAAHALFNYSISSRLSPKVWVASEAWLTSDLVMGLPGMAQMGTVLGFLQRGAQLHEFPQYVKTHLALATDPAFCSALGEREQGLEEDVVGQRCPQCDCITLQNVSAGLNHHQTFSVYAAVYSVAQALHNTLQCNASGCPAQDPVKPWQLLENMYNLTFHVGGLPLRFDSSGNVDMEYDLKLWVWQGSVPRLHDVGRFNGSLRTERLKIRWHTSDNQKPVSRCSRQCQEGQVRRVKGFHSCCYDCVDCEAGSYRQNPDDIACTFCGQDEWSPERSTRCFRRRSR FLAWGEPAVLLLLLLLSLALGLVLAALGLFVHHRDSPLVQASGGPLACFGLVCLGLVCLSVLLFPGQPSPARCLAQQPLSHLPLTGCLSTLFLQAAEIFVESELPLSWADRLSGCL RGPWAWLVVLLAMLVEVALCTWYLVAFPPEVVTDWHMLPTEALVHCRTRSWVSFGLAHATNATLAFLCFLGTFLVRSQPGCYNRARGLTFAMLAYFITWVSFVPLLANVQVVLRPAVQMGALLLCVLGILAAFHLPRCYLLMRQPGLNTPEFFLGGGPGDAQGQND GNTGNQGKHE

[0061] SEQ ID NO: 2, TAS1 / R2 receptor, UNIPROT Q8TE23, Homo sapiens, 839 amino acids (aa)MGPRAKTISSLFFLLWVLAEPAENSDFYLPGDYLLGGLFSLHANMKGIVHLNFLQVPMCKEYEVKVIGYNLMQAMRFAVEEINNDSSLLPGVLLGYEIVDVCYISNNVQPVLYFLAHEDNLLPIQEDYSNYISRVVAVIGPDNSESVMTVANFLSLFLLPQITYSAISDELRDKVRFPALLRTTPSADHHIEAMVQLMLHFRWNWIIVLVSSDTYGRDNGQLLGERVARRDICIAFQETLPTLQPNQNMTSEERQRLVTIVDKLQQSTARVVVVFSPDLTLYHFFNEVLRQNFTGAVWIASESWAIDPVLHNLTELRHLGTFLGITIQSVPIPGFSEFREWGPQAGPPPLSRTSQSYTCNQECDNCLNATLSFNTILRLSGERVVYSVYSAVYAVAHALHSLLGCDKSTCTKRVVYPWQLLEEIWKVNFTLLDHQIFFDPQGDVALHLEIVQWQWDRS QNPFQSVASYYPLQRQLKNIQDISWHTINNTIPMSMCSKRCQSGQKKKPVGIHVCCFECIDCLPGTFLNHTEDEYECQACPNNEWSYQSETSCFKRQLVFLEWHEAPTIAVALLAALGFLSTLAILVIFWRHFQTPIVRSAGGPMCFLMLTLLLVAYMVVPVYVGPPKVST CLCRQALFPLCFTICISCIAVRSFQIVCAFKMASRFPRAYSYWVRYQGPYVSMAFITVLKMVIVVIGMLATGLSPTTRTDPDDPKITIVSCNPNYRNSLLFNTSLDLLLSVVGFSFAYMGKELPTNYNEAKFITLSMTFYFTSSVSLCTFMSAYSGVLVTIVDLLVTVLNLLAIS LGYFGPKCYMILFYPERNTPAYFNSMIQGYTMRRD

[0062] In some embodiments, the agonist of the TAS1 / R3 and / or TAS1 / R2 receptor comprises a sweet protein. Sweet proteins are polypeptides, often isolated from fruits, that can bind to sweet taste receptors and elicit a sweet perception that can be 100 to 1,000 times greater than common ligands such as glucose. For additional information about sweet proteins, see e.g., Ohtsu et al. 2014, “Diverse signaling systems activated by the sweet taste receptor in human GLP-1 -secreting cells,” Mol Cell Endocrinol 394(1-2): 70-9 (2014); Sukumaram et at. 2022, “Sweet Taste Signaling: The Core Pathways and Regulatory Mechanisms,” Int J Mol Sci 23(15): 8225 (2022); Yamamoto et al. 2020, “Sweet proteins lysozyme and thaumatin are protein-type agonists for the calcium-sensing receptor,” Biochem Biophys Res Commun 521(1): 227-231 (2020); Jang et al. 2007, “Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide- 1,” PNAS 104(38): 15069-74 (2007); the contents of each of which are incorporated herein by reference in their entireties.

[0063] In some embodiments, the probiotic microbe is engineered to secrete 1, 2, 3, 4, 5, 6, 7, or more sweet proteins. In some embodiments, the sweet protein is selected from the group consisting of monellin (from serendipity berry), brazzein (from oubli fruit), thaumatin (from katemfe fruit), curculin (from fruit of Curculingo latifolia), mabinlin (from seeds of the mabinlag plant), miraculin (from miracle fruit), and pentadin (from oubli fruit), or variants thereof. In some embodiments, the sweet protein is soluble, e.g., in an aqueous solution. For example, in some embodiments, the sweet protein comprises soluble thaumatin I or soluble thaumatin II. In some embodiments, the sweet protein is truncated and / or comprises a functional fragment of the full-length sweet protein; such truncations and / or fragments can be easier to produce and / or sweeter than the full-length sweet protein. For example, in some embodiments, the sweet protein comprises a truncated form of brazzein (e.g., missing the cyclic first amino acid). In some embodiments, the sweet protein is engineered to have increased sweetness and / or potency, e.g., using amino acid substitutions. In some embodiments, the probiotic microbe is engineered to secrete a combination of sweet proteins selected from Table 2.

[0064] Table 2: Exemplary Sweet Protein Combinations

[0065] In some embodiments, the sweet protein comprises a single-chain polypeptide version of monellin. Monellin is a sweet protein that was isolated from the fruit of Dioscoreophyllum cumminsii. which is known as the serendipity berry and is native to West Africa. In its natural form, monellin is composed of two separate chains A and B. MNEI is a single-chain monellin in which chain B and chain A of natural monellin are fused C-terminusto N-terminus, respectively, via a Gly-Phe dipeptide linker. In some embodiments, MNEI comprises SEQ ID NO: 3 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to SEQ ID NO: 3

[0066] SEQ ID NO: 3, MNEI sequence (not including the N-terminal secretion signals, which are specific to the secretion system of the host and are cleaved off in the mature polypeptide): GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNKVIRPCMKKTIYENEGFREIKGY EYQLYVYASDKLFRADISEDYKTRGRKLLRFNGPVPPP

[0067] In some embodiments, the sweet protein is comprised by a chimeric polypeptide, e.g., fused to a carrier protein, such as an Fc fragment. In some embodiments, the probiotic microbe expresses a fusion protein which comprises MNEI or a fusion protein that comprises SEQ ID NO: 3 or comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to SEQ ID NO: 3, which maintains its function (e.g., sweetness, agonist of TAS1 / R3 and / or TAS1 / R2).

[0068] In some embodiments, the probiotic microbe expresses at least one sweet protein (e.g., MNEI, e.g., SEQ ID NO: 3) and at least one agonist or antagonist selected from Table 1

[0069] In some embodiments, the enteroendocrine modulating polypeptide comprises a hormone. In some embodiments, the hormone binds to a receptor on an enteroendocrine cell. In some embodiments, the hormone is naturally secreted by an enteroendocrine cell. In some embodiments, the hormone is selected from the group consisting of cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), glucagon- like peptide 2 (GLP-2), glicentin, oxyntomodulin, Peptide YY (PYY), secretin, Substance P (SP), xenin, glucose-dependent insulinotropic polypeptide (GIP), neurotensin, motilin, Gcg (GLP1 precursor), ghrelin, somatostatin, gastrin, oxyntomodulin (OXM), and b-casomorphin, or variants thereof (e.g., cleaved, glycosylated variants of the hormones). In some embodiments, the enteroendocrine modulating polypeptide modulates the ratios of variants of a hormone (e.g., cleaved, glycosylated variants of the hormones). As a non-limiting example, the enteroendocrinemodulating polypeptide can modulate the ratio of active and inactive (e.g., degraded) forms or variants of a hormone, such as GLP-1 or GLP-2.

[0070] In some embodiments, enteroendocrine modulating polypeptide modulates the level or activity of a neurotransmitter, such as serotonin.

[0071] In some embodiments, the enteroendocrine modulating polypeptide comprises an antagonist of Neuropeptide Y Receptor Y1 (NpylR). In some embodiments, the enteroendocrine modulating polypeptide comprises an antagonist of dipeptidyl peptidase IV (DPP-IV) (see e.g. Table 1).

[0072] In some embodiments, the nucleic acid sequence encoding the enteroendocrine modulating polypeptide is codon-optimized for expression in the probiotic microbe. In some embodiments, the nucleic acid sequence encoding the enteroendocrine modulating polypeptide is codon-optimized for expression in a yeast. In some embodiments, the nucleic acid sequence encoding the enteroendocrine modulating polypeptide is codon-optimized for expression in a bacterium.

[0073] In some embodiments, the enteroendocrine modulating polypeptide comprises a binding domain, such as an antigen-binding domain, that specifically binds at least one receptor expressed on an enteroendocrine cell. Non-limiting examples of enteroendocrine receptors are provided herein. In some embodiments, the receptor is a G-protein-coupled receptor expressed on an enteroendocrine cell. In some embodiments, such binding sterically blocks the activation of the receptor on the enteroendocrine cell. In some embodiments, such binding increases a specific conformation state (e.g, active or inactive) of the receptor. In some embodiments, the enteroendocrine modulating polypeptide comprises an antibody or fragment thereof, a nanobody (e.g, camelid antibody fragments, single domain-based VHHs), or a single-chain variable fragments (scFv, protein fusions of the variable regions of heavy and light chains of immunoglobulins).

[0074] In some embodiments, the enteroendocrine modulating polypeptide is selected from the group consisting of: Mogamulizumab (KYOWA HAKKO KIRIN), PRO 140 (CYTODYN), Erenumab (AMGEN / NOVARTIS), Ulocuplumab (BRISTOL-MYERS SQUIBB), Plozalizumab (MILLENNIUM PHARMACEUTIC ALS / TAKEDA ONCOLOGY), REMD-477 (AMGEN / REMD BIOTHERAPEUTICS), IPH5401 (INNATE PHARMA), BI 665088 (ABLYNX / BOEHRINGER INGELHEIM), Vantictumab (ONCOMED / BAYER), FZD8-IgG (ONCOMED), OTSA-101-DTPA-90Y (UNIVERSITY OF TOKYO / CENTRE LEON-BERARD), OMP-131R10 (ONCOMED), anti-CXCR2biparatopic nanobody (ABLYNX / NOVARTIS), BNC-101 (BIONOMICS), GMA102 (GMAX BIOPHARM), GMA105 (GMAX BIOPHARM), Namacizumab (BIRD ROCK BIO), BNC-102 (bispecific) (BIONOMICS), MCLA-158 (bispecific) (MERUS), anti-FZD2 antibody (HARVARD UNIVERSITY), EDD 7H9 (EXPRESSION DRUG DESIGNS), anti- ELTD1 antibody (UNIVERSITY OF OKLAHOMA HEALTH SCIENCES CENTER), JT07 (JYANT TECHNOLOGIES), JTO8 (JYANT TECHNOLOGIES), anti-CRTH2 antibody (GENENTECH), P-007 (NB HEALTH LABORATORY), and anti-CCR9 antibody (CENTRO NACIONAL DE BIOTECNOLOGIA). See e g., Steyaert et al. “Nanobody stabilization of G protein coupled receptor conformational states,” Curr Opin Struct Biol. 21(4): 567-572 (2011); Hutchings et al. “Opportunities for therapeutic antibodies directed at G-protein-coupled receptors,” Nat Rev Drug Discov 16(9): 787-810 (2017); the contents of each of which are incorporated herein by reference in their entireties.Nucleic Acids and Vectors

[0075] The enteroendocrine modulating polypeptides described herein can be encoded and / or expressed by nucleic acids and / or vectors. Accordingly, in one aspect described herein is a nucleic acid encoding and / or capable of expressing an enteroendocrine modulating polypeptide described herein. In another aspect, described herein is a vector comprising such a nucleic acid. In another aspect, described herein is a vector encoding and capable of expressing an enteroendocrine modulating polypeptide.

[0076] In one aspect, described herein is a nucleic acid encoding and capable of expressing an enteroendocrine modulating polypeptide, wherein the enteroendocrine modulating polypeptide is codon-optimized for expression in a yeast and / or bacterium, and / or the enteroendocrine modulating polypeptide is operably linked to a yeast and / or bacterial regulatory element.

[0077] In one aspect, described herein is a nucleic acid encoding and capable of expressing an enteroendocrine modulating polypeptide, wherein the enteroendocrine modulating polypeptide is codon-optimized for expression in a yeast, and the enteroendocrine modulating polypeptide is operably linked to a yeast regulatory element.

[0078] In one aspect, described herein is a nucleic acid encoding and capable of expressing an enteroendocrine modulating polypeptide, wherein the enteroendocrine modulating polypeptide is codon-optimized for expression in a bacterium, and the enteroendocrine modulating polypeptide is operably linked to a bacterial regulatory element.

[0079] In some embodiments of any of the aspects, the nucleic acid as described herein comprises DNA. In some embodiments of any of the aspects, the nucleic acid as described herein consists essentially of DNA. In some embodiments of any of the aspects, the nucleic acid as described herein consists of DNA.

[0080] In some embodiments of any of the aspects, the nucleic acid as described herein comprises RNA. In some embodiments of any of the aspects, the nucleic acid as described herein consists essentially of RNA. In some embodiments of any of the aspects, the nucleic acid as described herein consists of RNA.

[0081] A nucleic acid molecule that encodes an enteroendocrine modulating polypeptide as described herein can be introduced into a cell or cells using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule encoding an enteroendocrine modulating polypeptide as described herein can also be accomplished by integrating the nucleic acid molecule into the genome.

[0082] In some embodiments, one or more of the enteroendocrine modulating polypeptides described herein is expressed in a recombinant expression vector or plasmid. As used herein, the term “vector” refers to a polynucleotide sequence suitable for transferring nucleic acids (e.g., a nucleic acid encoding an enteroendocrine modulating polypeptide as described herein) into a host cell, such as a probiotic microbe. The vector can encompass any genetic element that is capable of replication when associated with the proper control elements and that can transfer nucleic acid sequences to cells. The term “vector” includes a plasmid, a cloning vector, an expression vector, naked DNA, a mini-chromosome, a chromosome, a transposon, a cosmid, a virus, virion, phage, and the like. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Press (1989). One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, wherein additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vector. However, the technology is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0083] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a nucleic acid encoding an enteroendocrine modulating polypeptide operably linked to at least one heterologous genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like). In some embodiments of any of the aspects, the vector comprises the 2-micron circle plasmid.

[0084] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that the altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild- type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell.

[0085] A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector can be cut in a determinable fashion and into which a desired DNA sequence (e.g., a nucleic acid encoding an enteroendocrine modulating polypeptide as described herein) can be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence can occur many times as the plasmid increases in copy number within the host cellsuch as a host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication can occur actively during a lytic phase or passively during a lysogenic phase.

[0086] An expression vector is one into which a desired DNA sequence (e.g., a nucleic acid encoding an enteroendocrine modulating polypeptide as described herein) can be inserted by restriction and ligation such that it is operably joined to regulatory sequences and can be expressed as an RNA transcript. Vectors can further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds (e.g., ampicillin resistance), genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., P-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). In certain embodiments, the vectors used herein are capable of autonomous replication and expression of the enteroendocrine modulating polypeptide present in the DNA segments to which they are operably joined. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector can comprise additional elements, for example, the expression vector can have two replication systems, thus allowing it to be maintained in two organisms, for example in fungal cells for expression and in a prokaryotic host for cloning and amplification.

[0087] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (or RNA). That heterologous DNA (or RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.

[0088] As used herein, the term “viral vector” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the enteroendocrine modulating polypeptide coding sequence in place of non-essential viral genes. The vector and / or particle can be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo.Numerous forms of viral vectors are known in the art. Non-limiting examples of a viral vector of this invention include an AAV vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpesvirus vector, an alphavirus vector, a poxvirus vector, a baculovirus vector, and a chimeric virus vector.

[0089] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleic acid of interest (e.g., a nucleic acid encoding an enteroendocrine modulating polypeptide as described herein) in the probiotic microbe in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.Regulatory Elements

[0090] In some embodiments, the nucleic acid encoding and capable of expressing an enteroendocrine modulating polypeptide is operably linked to at least one regulatory element permitting expression of the enteroendocrine modulating polypeptide in a host cell, such as a yeast cell or a bacterial cell. In some embodiments, expression includes, but is not limited to, transcription, splicing, translation, post-translational processing, and secretion of the bacterial polypeptide coding sequence or encoded bacterial polypeptide in the host cell.

[0091] In some embodiments, the regulatory element comprises at least one yeast-derived expression control element, which directs or permits expression of the enteroendocrine modulating polypeptide in a yeast host cell. In some embodiments, the at least one yeast- derived expression control element is operably linked to the enteroendocrine modulating polypeptide coding sequence. In some embodiments, the at least one yeast-derived expression control element comprises a yeast-derived promoter. In some embodiments, the at least one yeast-derived expression control element comprises a yeast-derived secretion signal. In some embodiments, the secretion signal comprises or is derived from the secretion of signal of a yeast alpha mating factor.

[0092] When the nucleic acid molecule that encodes and is capable of expressing an enteroendocrine modulating polypeptide described herein is expressed in a probiotic microbe, a variety of transcription control sequences (e.g., promoter / enhancer sequences) can be used to direct its expression. The promoter can be a promoter native to the probiotic microbe. The promoter can be a promoter derived from another species that is functional in the probiotic microbe. In some embodiments the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of the enteroendocrine modulatingpolypeptide coding sequence in the probiotic microbe. A variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule or condition (e.g., oxygen concentration, pH, presence of cell-free DNA / RNA, specific nutritional sources (e.g., sugars, fibers), starvation conditions, temperature, etc.). In some embodiments, the promoter comprises the tdh3 (GDP) promoter.

[0093] The precise nature of the regulatory sequences needed for expression can vary between species or cell types, but in general can include, as necessary, 5' non-transcrib ed and 5' non-translated sequences involved with the initiation of transcription and translation respectively. In particular, such 5' non-transcribed regulatory sequences can include a promoter region which includes a promoter sequence for transcriptional control of the enteroendocrine modulating polypeptide coding sequence. Regulatory sequences can also include enhancer sequences or upstream activator sequences as desired.

[0094] As used herein, the enteroendocrine modulating polypeptide coding sequence and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the enteroendocrine modulating polypeptide under the influence or control of the regulatory sequences. If it is desired that at least one enteroendocrine modulating polypeptide coding sequence be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the enteroendocrine modulating polypeptide coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the enteroendocrine modulating polypeptide coding sequence, or (3) interfere with the ability of the enteroendocrine modulating polypeptide coding sequence to be translated into a protein.Probiotic Microbes

[0095] Described herein in multiple aspects are probiotic microbes engineered to secrete at least one enteroendocrine modulating polypeptide as described herein. In some embodiments, the probiotic microbe comprises a nucleic acid encoding and capable of expressing an enteroendocrine modulating polypeptide as described herein. In some embodiments, the probiotic microbe comprises a vector encoding and capable of expressing an enteroendocrine modulating polypeptide as described herein.

[0096] In some embodiments, the probiotic microbe is capable of expressing the enteroendocrine modulating polypeptide. In some embodiments, the nucleic acid comprisesregulatory elements permitting expression of the enteroendocrine modulating polypeptide in the probiotic microbe.

[0097] In some embodiments, the probiotic microbe is capable of transcribing, splicing, and translating the enteroendocrine modulating polypeptide. In some embodiments, the probiotic microbe is capable of splicing at least one intron out of an RNA encoding the enteroendocrine modulating polypeptide. In some embodiments, the probiotic microbe is capable of post-translational processing of the encoded enteroendocrine modulating polypeptide. In some embodiments, the probiotic microbe is capable of secreting the enteroendocrine modulating polypeptide.

[0098] In some embodiments, the probiotic microbe is a fungus. In some embodiments, the probiotic microbe is a yeast. In some embodiments, the probiotic microbe is a budding yeast. In some embodiments, the probiotic microbe is a hemiascomycetous yeast.

[0099] In some embodiments, the probiotic microbe belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Debaryomyces, Candida, and Yarrowia. In some embodiments, the probiotic microbe belongs to the Saccharomyces genus. In some embodiments, the probiotic microbe belongs to a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Debaryomyces, Candida, Yarrowia, Kazachstania, Lachancea, Hansenula, Malassezia, Cryptococcus, Rhodotorula, Schizosaccharomyces, Komagataella, Meyerozyma, Hanseniaspora, Rhizophagus, and Ogataea.

[0100] In some embodiments, the probiotic microbe is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces unisporus, Saccharomyces servazzii (also referred to as Kazachstania servazzii), Saccharomyces kluyveri (also referred to as Lachancea kluyveri), Kluyveromyces marxianus, Pichia angusta (also referred to as Hansenula polymorpha or Ogataea polymorpha), Debaryomyces hansenii (also referred to as Candida famata), Candida tropicalis, Yarrowia lipolytica, Kluyveromyces lactis, Candida albicans, Candida glabrata, Candida krusei, Candida auris, Lachancea thermotolerans, Malassezia furfur, Malassezia globosa, Malassezia pachydermatis, Cryptococcus neoformans, Rhodotorula rubra, Rhodotorula glutinis, Schizosaccharomyces pombe, Komagataella pastoris, Pichia guilliermondii, Meyerozyma gruessi, Hanseniaspora osmophila, and Rhizophagus irregularis. In some embodiments, the probiotic microbe is selected from the group consisting of Saccharomyces cerevisiae (S. cerevisiae), Saccharomyces boulardii (S. boulardii), and Saccharomyces unisporus (S. unisporus). Insome embodiments, the probiotic microbe is Saccharomyces cerevisiae. In some embodiments, the probiotic microbe is Saccharomyces boulardii.

[0101] In some embodiments, the probiotic microbe is a bacterium. Non-limiting examples of probiotic bacteria belong to a genus selected from Lactobacillus, Pediococcus, Streptococcus, Bacillus, Enter occous, Escherichia, Vibrio, Bacteroides, and Bifidobacterium. In some embodiments, the probiotic microbe is a bacterial species selected from the group consisting of: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus salivarius, Pediococcus pentosaceus, Streptococcus thermophiles, Bacillus subtilis, Bacillus coagulans, Enteroccous faecium, Escherichia coli (e.g., Nissle), Vibrio natriegens, Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium asteroids, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum.

[0102] In some embodiments, the probiotic microbe is dried and viable. In some embodiments, the probiotic microbe is freeze-dried. In some embodiments, the probiotic microbe is spray-dried. In some embodiments, the viable, dried probiotic microbe is minimally metabolically active when in the dried state. In some embodiments, the viable, dried probiotic microbe becomes metabolically active once reconstituted in an aqueous liquid.

[0103] In some embodiments, the probiotic microbe is capable of sporulation. Accordingly, in some embodiments the probiotic microbe is in a spore form.

[0104] In some embodiments, the probiotic microbe is capable of colonizing the gastrointestinal tract. As used herein in regard to probiotic microbes, the term “colonization” refers to the presence of a microbe on / in a host (e.g., within the gut), with growth and multiplication of the microbe, but without interaction between host and microbe (e.g., no immune response).

[0105] In some embodiments, the probiotic microbe comprises an off-switch or a suicide trigger to remove the microbe from the gastrointestinal system and / or to provide regulation of the secretion of the enteroendocrine modulating polypeptide by the probiotic microbe.Compositions

[0106] In one aspect, described herein is a composition comprising a probiotic microbe as described herein, formulated for delivery to a subject. In one aspect, described herein is acomposition comprising a nucleic acid as described herein, formulated for delivery to a subject. In one aspect, described herein is a composition comprising a vector as described herein, formulated for delivery to a subject.

[0107] In one aspect, described herein is a formulation comprising the probiotic microbe as described herein and a therapeutically acceptable carrier. The formulation can, for example, deliver the probiotic microbe as a probiotic to the gut, and can comprise, for example, one or more carriers. In one aspect, described herein is a pharmaceutical composition comprising the at least one probiotic microbe as described herein and a pharmaceutically acceptable carrier.

[0108] In some embodiments of any of the aspects, the probiotic microbe is formulated in a food composition. In some embodiments of any of the aspects, the food composition comprises a yogurt or a yogurt beverage. In some embodiments of any of the aspects, the food composition comprises a water-containing product (aqueous product), a baked good, a confectionary product (e.g., chocolate), candy, a gel, an ice cream, a cereal, a restored fruit product, a snack bar, a food bar, a breakfast cereal bar, a pasty food product, a flavoring paste, a sauce, a dip, a milk-product (e.g., yogurt, cheese), kefir, a beverage, a drink additive, milk, milk powder, a fruit juice, a foodstuff additive (e.g., protein sprinkles), or a dietary supplement product (e.g., a tablet, capsule).

[0109] In some embodiments of any of the aspects, the probiotic microbe is formulated in a medical food. As used herein, “medical food” is understood to mean a food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation. In some embodiments of any of the aspects, the probiotic microbe is formulated in a supplement.

[0110] In one aspect, described herein is a food composition comprising the probiotic microbe as described herein. In one aspect, described herein is a medical food comprising the probiotic microbe as described herein. In one aspect, described herein is a supplement comprising the probiotic microbe as described herein. In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a probiotic microbe as described herein, and optionally a pharmaceutically acceptable carrier.

[0111] In some embodiments, the active ingredients of the pharmaceutical composition comprise the enteroendocrine modulating polypeptide as described herein. In someembodiments, the active ingredients of the pharmaceutical composition consist essentially of the enteroendocrine modulating polypeptide as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of enteroendocrine modulating polypeptide as described herein.

[0112] In some embodiments, the active ingredients of the pharmaceutical composition comprise the probiotic microbe as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of the probiotic microbe as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of the probiotic microbe as described herein.

[0113] Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum component, such as serum albumin, HDL and LDL; (24) C2-C12 alcohols; and (25) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g., the enteroendocrine modulating polypeptide and / or the probiotic microbe as described herein.

[0114] Pharmaceutical compositions comprising a probiotic microbe as described herein can be formulated to be suitable for oral administration, for example as discrete dosageforms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the probiotic microbe as described herein, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 2lst Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0115] In some embodiments, the probiotic microbe secretes the enteroendocrine modulating polypeptide in the stomach, duodenumjejunum, ileum, cecum, mid-colon, and / or distal colon of a subject. In some embodiments, the probiotic microbe secretes the enteroendocrine modulating polypeptide in the distal colon of a subject. Such a secretion can be beneficial as the majority of dietary components are absorbed in the small intestine before reaching the colon, and there are few components left in the intra-intestinal fluids to stimulate enteroendocrine cells in the colon, especially the distal colon.

[0116] In some embodiments, the probiotic microbe is formulated for delivery to the gastrointestinal system. In some embodiments, the probiotic microbe is encapsulated. In some embodiments, the encapsulation comprises an enteric coating, for example, which can permit the release of the probiotic microbe in the duodenum ejunum, ileum, cecum, mid-colon, and / or distal colon. In some embodiments, the probiotic microbe is released in the distal colon.

[0117] In some embodiments, the composition further comprises at least one prebiotic, for example, 1, 2, 3, 4, 5, or more prebiotics. In some embodiments, the prebiotic supports growth and / or colonization by the probiotic microbe in the gut of a mammalian subject. Nonlimiting examples of such prebiotics include: an amino acid (e.g., arginine, glutarate, and ornithine), biotin, fructooligosaccharide, galactooligosaccharide, hemi cellulose (e.g., arabinoxylan, xylan, xyloglucan, glucomannan), inulin, chitin, lactulose, mannan oligosaccharide, oligofructose-enriched inulin, gum (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectin (e.g., xylogal actouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soy fiber, sugarbeet fiber, pea fiber, com bran, and oat fiber), xylooligosaccharide, and polyamine (e.g., spermidine, putrescine).Dosing

[0118] In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) to a subject in order to prevent or alleviate a symptom of a disease or disorder. As used herein, “alleviating a symptom of a disease or disorder” is ameliorating any condition or symptom associated with the disease or disorder. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art.

[0119] Subjects can be administered an effective (e.g., prophylactic and / or therapeutic) amount of a composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) comprising a probiotic microbe as described herein, such as, about 101colony forming units (CFUs), about 102CFUs, about 103CFUs, about 104CFUs, about 105CFUs, about 106CFUs, about 107CFUs, about 108CFUs, about 109CFUs, about 1010CFUs, about 1011CFUs, about 1012CFUs, about 1013CFUs, or more of the probiotic microbe as described herein. In one embodiment, the subject is administered about 108CFUs of the probiotic microbe as described herein.

[0120] In embodiments related to compositions such as food compositions, medical foods, supplements, and / or probiotics, the subject can be administered a predetermined serving size. As non-limiting examples, the serving size can be about 1 mL, about 5 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, about 100 mL, or more, about 1 g, about 5 g, about 10 g, about 20 g, about 30 g, about 40 g, about 50 g, about 60 g, about 70 g, about 80 g, about 90 g, about 100 g, or more, or 1 pill, 2 pills, 3 pills, 4 pills, 5 pills, or more.

[0121] The term “effective amount” as used herein refers to the amount of the probiotic microbe as described herein needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) to provide the desired effect. As used herein, the phrase “effective amount” means an amount sufficient to achieve a meaningful benefit (including, but not limited to, e.g., metabolic energy homeostasis modulation).

[0122] It is understood that for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using routine experimentation. Given theintricacies of the body and the nature of cell establishment, the “effective amount” of cells can vary among different patients; however, one can easily determine in hindsight if the amount of cells administered was indeed an “effective amount.” Thus, further treatments can be modified accordingly. Note that long-term colonization or establishment, while often desirable, is not necessary for effective treatment as regular administration can achieve effective treatment as well.

[0123] The term “therapeutically effective amount” therefore refers to an amount of the probiotic microbe as described herein that is sufficient to provide a particular therapeutic effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.

[0124] Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[0125] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. A dose can be formulated in animal models. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the administration.

[0126] The dosage ranges for the administration of the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition), according to the methods described herein depend upon, for example, the form of the composition, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced. The dosage should not be so large as to cause adverse side effects, such as yeast infection. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by oneof skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0127] The efficacy of the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) in, e.g., the prevention and / or treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, an administration is considered “effective,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following administration according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition according to the methods described herein or any other measurable parameter appropriate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein.

[0128] Prevention keeps a subject from developing a disease or disorder or condition, such as a metabolic disease, and includes prophylactic administration. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms; or (2) relieving the severity of the disease, e.g., causing regression of symptoms. Prevention or treatment can also include delaying the onset of a disease or condition in the subject.

[0129] An effective amount for the prevention or treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective prevention or treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein. When using an experimental animal model, efficacy of prevention or treatment is evidenced when a statistically significant change in a marker is observed. In vitro and animal model assays allow the assessment of a given dose of the composition.

[0130] With respect to duration and frequency of administration, it is typical for skilled clinicians to monitor subjects in order to determine when the administration is providing a benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue administration, resume administration, or make other alterations to the administration regimen.

[0131] In certain embodiments, an effective dose of a composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) can be administered to a subject once. In certain embodiments, an effective dose of a composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) can be administered to a subject repeatedly.

[0132] The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject’s sensitivity to the probiotic microbe. The desired dose or amount can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be one or more doses and / or administrations daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition) can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

[0133] In some embodiments, after an initial regimen, the doses and / or treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer.Administration

[0134] A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral or intrarectal.

[0135] In some embodiments of any of the aspects, the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, apharmaceutical composition) as described herein is administered as a monotherapy, e.g., another treatment for the disease or disorder is not administered to the subject.

[0136] The methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g., as part of a combinatorial therapy.

[0137] By way of non-limiting example, if a microbial infection is to be prevented or treated in a subject according to the methods described herein, the subject can also be administered at least one antimicrobial agent effective against the specific microbe of the microbial infection, in addition to the composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition).

[0138] As used herein, the term “antimicrobial agent” (also referred to herein as an antimicrobial, antimicrobial therapeutic, antibiotic and the like) refers to a molecule or composition which destroys microbes (i.e., bacteria, fungi, viruses, parasites, and / or microbial spores) or prevents or inhibits their development, proliferation and / or pathogenic action. The term “antimicrobial” thus comprises antibacterials, antifungals, and antivirals. Exemplary antimicrobial agents include, but are not limited to, small organic or inorganic molecules; peptides; proteins; peptide analogs and derivatives; peptidomimetics; antibodies (polyclonal or monoclonal) that target a microbe; antigen binding fragments of such antibodies; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. In one embodiment, the antimicrobial agent does not kill the probiotic microbe as described herein. In another embodiment, the antimicrobial agent is administered before administering a probiotic microbe as described herein. In another embodiment, administration of the antimicrobial agent is halted prior to administration of the probiotic microbe a described herein.

[0139] In some embodiments of any of the aspects, the antimicrobial agent can be selected from aminoglycosides, ansamycins, beta-lactams, bis-biguanides, carbacephems, carbapenems, cationic polypeptides, cephalosporins, fluoroquinolones, glycopeptides, iron- sequestering glycoproteins, linosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quaternary ammonium compounds, quinolones, silver compounds, sulfonamides, tetracyclines, and any combinations thereof. In some embodiments of any of the aspects, the antimicrobial agent can comprise an antibiotic.

[0140] Some exemplary specific antimicrobial agents include broad penicillins, amoxicillin (e.g., Ampicillin, Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin, Ticarcillin), Penicillins and Beta Lactamase Inhibitors (e.g., Amoxicillin-Clavulanic Acid, Ampicillin-Sulbactam, Benzylpenicillin, Cioxacillin, Dicloxacillin, Methicillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin Tazobactam, Ticarcillin Clavulanic Acid, Nafcillin), Cephalosporins (e.g., Cephalosporin I Generation, Cefadroxil, Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine), Cephalosporin II Generation (e.g., Cefaclor, Cefamandole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil, Cefmetazole, Cefuroxime, Loracarbef), Cephalosporin III Generation (e.g., Cefdinir, Ceftibuten, Cefoperazone, Cefixime, Cefotaxime, Cefpodoxime proxetil, Ceftazidime, Ceftizoxime, Ceftriaxone), Cephalosporin IV Generation (e.g., Cefepime), Macrolides and Lincosamides (e.g., Azithromycin, Clarithromycin, Clindamycin, Dirithromycin, Erythromycin, Lincomycin, Troleandomycin), Quinolones and Fluoroquinolones (e.g., Cinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid, Gemifloxacin, Perfloxacin), Carbapenems (e.g., Imipenem-Cilastatin, Meropenem), Monobactams (e.g., Aztreonam), Aminoglycosides (e.g., Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin), Glycopeptides (e.g., Teicoplanin, Vancomycin), Tetracyclines (e.g., Demeclocycline, Doxycycline, Methacycline, Minocycline, Oxytetracycline, Tetracycline, Chlortetracycline), Sulfonamides (e.g., Mafenide, Silver Sulfadiazine, Sulfacetamide, Sulfadiazine, Sulfamethoxazole, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, Sulfamethizole), Rifampin (e.g., Rifabutin, Rifampin, Rifapentine), Oxazolidinones (e.g., Linezolid, Streptogramins, Quinupristin Dalfopristin), Bacitracin, Chloramphenicol, Fosfomycin, Isoniazid, Methenamine, Metronidazole, Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin, Polymyxin, Spectinomycin, Trimethoprim, Colistin, Cycloserine, Capreomycin, Ethionamide, Pyrazinamide, Para-aminosalicylic acid, Erythromycin ethyl succinate, and the like.

[0141] In some embodiments of any of the aspects, the antifungal is selected from the group consisting of polyene antifungals, Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, imidazole antifungals, triazole antifungals, thiazole antifungals, Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Triazolesfedit], Albaconazole, Efinaconazole, Epoxiconazole,Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Propi conazole, Ravuconazole, Terconazole, Voriconazole, Abafungin, Allylamines, amorolfin, butenafine, naftifine, terbinafine, Echinocandins, Anidulafungin, Caspofungin, Micafungin, Aurones, Benzoic acid, Ciclopirox, Flucytosine, 5-fluorocytosin, Griseofulvin, Haloprogin, Tolnaftate, Undecylenic acid, Triacetin, Crystal violet, Castellani’s paint, Orotomide, Miltefosine, Potassium iodide, Coal tar, Copper(II) sulfate, Selenium disulfide, Sodium thiosulfate, Piroctone olamine, lodoquinol, clioquinol, Acrisorcin, Zinc pyrithione, and Sulfur. Additional antifungals known in the art can also be used.

[0142] In some embodiments of any of the aspects, the antiviral is selected from the group consisting of: Abacavir, Acyclovir, Adefovir, Amantadine, Ampligen, Amprenavir, antiretroviral, Arbidol, Atazanavir, Atripla, Cidofovir, Combivir, Darunavir, Delavirdine, Didanosine, Docosanol, Dolutegravir, Ecoliever, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Famciclovir, Fomivirsen, Fosamprenavir, Foscamet, Fosfonet, Fusion inhibitor, Ibacitabine, Idoxuridine, Imiquimod, Imunovir, Indinavir, Inosine, Integrase inhibitor, Interferon, Interferon type I, Interferon type II, Interferon type III, Lamivudine, Lopinavir, Loviride, Maraviroc, Methisazone, Moroxydine, Nelfinavir, Nevirapine, Nexavir, Nitazoxanide, Norvir, Nucleoside analogues, Oseltamivir (Tamiflu), Peginterferon alfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Protease inhibitor, Pyramidine, Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rimantadine, Ritonavir, Saquinavir, Sofosbuvir, Stavudine, Synergistic enhancer (antiretroviral), Telaprevir, Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex), Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir (Relenza), Zidovudine. Additional antivirals known in the art can also be used.

[0143] By way of another non-limiting example, if pain or inflammation is to be prevented or treated in a subject according to the methods described herein, the subject can also be administered a second agent and / or treatment known to be beneficial for subjects suffering from pain or inflammation. Examples of such agents and / or treatments include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs - such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e.g. cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclometasone); methotrexate; sulfasalazine; leflunomide; anti-TNF medications; cyclophosphamide; pro-resolving drugs; mycophenolate; or opiates (e.g. endorphins,enkephalins, and dynorphin), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like.

[0144] In some embodiments of any of the aspects, the subject is co-administered at least one prebiotic in addition to the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition). Nonlimiting examples of such prebiotics include amino acid (e.g., arginine, glutarate, and ornithine), biotin, fructooligosaccharide, galactooligosaccharide, hemi cellulose (e.g., arabinoxylan, xylan, xyloglucan, glucomannan), inulin, chitin, lactulose, mannan oligosaccharide, oligofructose-enriched inulin, gum (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectin (e.g., xylogal actouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soy fiber, sugarbeet fiber, pea fiber, com bran, and oat fiber), xylooligosaccharide, and polyamine (e.g., spermidine, putrescine).

[0145] In some embodiments of any of the aspects, the subject is co-administered at least one probiotic in addition to the composition (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition). In some embodiments of any of the aspects, the subject is co-administered with the composition at least one fungal probiotic. In some embodiments of any of the aspects, the subject is coadministered with the composition at least one bacterial probiotic.Methods of Use

[0146] In one aspect, described herein are methods of enteroendocrine modulation, modulating blood glucose homeostasis, and / or treating type II diabetes. In one aspect, the method comprises administering an enteroendocrine-modulating amount of a probiotic microbe as described herein, a composition as described herein, a pharmaceutical composition as described herein, a food composition as described herein, a medical food as described herein, or a supplement as described herein to a subject in need of enteroendocrine modulation.

[0147] In one aspect, described herein is a method of modulating blood glucose homeostasis. In one aspect, described herein is a method of treating type II diabetes. In some embodiments, the method comprises administering an enteroendocrine-modulating amount of a probiotic microbe engineered to secrete a polypeptide agonist of the T1R2-T1R3 sweet receptor. In some embodiments, the polypeptide agonist of the T1R2-T1R3 sweet receptor isselected from the group consisting of monellin, brazzein, thaumatin, curculin, mabinlin, miraculin, or pentadin, or any of the combinations shown in Table 2.

[0148] In some embodiments, the method comprises administering an enteroendocrine- modulating amount of a probiotic microbe engineered to secrete a polypeptide agonist of the GLP-1 receptor. In some embodiments of any one of the aspects, the probiotic microbe is engineered to secrete glucagon-like peptide 1 (GLP-1) and / or glucagon-like peptide 2 (GLP- 2).

[0149] In one aspect, described herein is a method of preventing or treating a disease in a subject in need thereof. In some embodiments, the method comprises administering to the subject an effective amount of probiotic microbe as described herein or a composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition).

[0150] The compositions described herein can be administered to a subject in need thereof, for instance for the prevention and / or treatment of a metabolic disease, including but not limited to diabetes (e.g., type II diabetes), obesity, or liver disease.

[0151] In one aspect, described herein is a method of promoting heath in a subject. In some embodiments, the method comprises administering to the subject an effective amount of a probiotic microbe as described herein or a composition as described herein (e.g., a food composition, a medical food, a supplement, a formulation, a probiotic composition, a pharmaceutical composition). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal.

[0152] In some embodiments of any of the aspects, the enteroendocrine modulating polypeptide secreted by the engineered probiotic microbe modulates metabolic energy homeostasis in the subject. Non-limiting examples of elements of metabolic energy homeostasis include modulation of glucose levels, or modulation of metabolism, modulation of type 2 diabetes, weight management, satiety, gastric emptying, gut motility, nutrient absorption, gut hormone signaling (e.g., CCK signaling), management of dyslipidemia (e.g., imbalance of lipids such as cholesterol, low-density lipoprotein cholesterol, (LDL-C), triglycerides, and high-density lipoprotein (HDL)), and the like.

[0153] In some embodiments of any of the aspects, the method of prevention or treatment can comprise first determining and / or diagnosing a subject or patient who can benefit from treatment by a composition described herein. In some embodiments, such determination and / or diagnosis comprises detecting or measuring an abnormal level of at least one guthormone (e.g., GLP-1, PYY, ghrelin), at least one metabolic marker (e.g., Brain-Derived Neurotrophic Factor (BDNF), C-peptide, glucagon, insulin, leptin, monocyte chemoattractant protein-1 (MCP-1), fibroblast growth factor 12 (FGF-12)) and / or at least one adipokine (e.g., IL-10, IL-lb, IL-6, TNF-a), see e.g., Fig. 3B.

[0154] In some embodiments, the subject has previously been determined to have an abnormal level of an analyte described herein relative to a reference. In some embodiments, the reference level can be the level in a sample of similar cell type, sample type, sample processing, and / or obtained from a subject of similar age, sex and other demographic parameters as the sample / subject. In some embodiments, the test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g., the same number and type of cells.

[0155] The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a blood or plasma sample from a subject. In some embodiments of any of the aspects, the technology described herein encompasses several examples of a biological sample. In some embodiments of any of the aspects, the biological sample is cells, or tissue, or peripheral blood, or bodily fluid. In some embodiments of any of the aspects, the biological sample comprises feces. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; semen; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and / or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any of the aspects, a test sample can comprise cells from a subject.

[0156] In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise performing or having performed an assay on a sample obtained from the subject to determine / measure the level of analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise ordering or requesting an assay on a sample obtained from the subject to determine / measure the level ofthe analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise receiving the results of an assay on a sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise receiving a report, results, or other means of identifying the subject as a subject with a decreased level of the analyte.

[0157] In one aspect of any of the embodiments, described herein is a method of preventing or treating a disease or disorder (e.g., a metabolic disease) in a subject in need thereof, the method comprising: a) determining if the subject has an abnormal level of an analyte described herein; and b) instructing or directing that the subject be administered a composition comprising a probiotic microbe as described herein if the level of the analyte is abnormal relative to a reference. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and / or treatment recommendations in view of the assay results.Definitions

[0158] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. 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. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0159] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal, e.g., for an individual without a given disorder.

[0160] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0161] As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0162] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a metabolic disease. A subject can be male or female.

[0163] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., a metabolic disease, e.g.,diabetes type II diabetes, obesity, or liver disease) or one or more complications related to such a condition, and optionally, have already undergone treatment for a metabolic disease or the one or more complications related to a metabolic disease. Alternatively, a subject can also be one who has not been previously diagnosed as having a metabolic disease or one or more complications related to a metabolic disease. For example, a subject can be one who exhibits one or more risk factors for a metabolic disease or one or more complications related to a metabolic disease or a subject who does not exhibit risk factors.

[0164] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0165] As used herein, the terms “protein” and “polypeptide” are used interchangeably to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0166] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0167] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested to confirm that a desired activity, e.g. binding to a receptor as described herein.

[0168] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into lie or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into lie; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0169] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a polypeptide which retains at least 50% of the wild-type reference polypeptide’s activity. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0170] In some embodiments, the polypeptide described herein can be a variant of a polypeptide sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acidsequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a protein or fragment thereof that retains activity of the native or reference polypeptide. A wide variety of, for example, PCR-based, site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan to generate and test artificial variants.

[0171] A variant amino acid or DNA sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).

[0172] A variant amino acid sequence can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to a native or reference sequence. As used herein, “similarity” refers to an identical amino acid or a conservatively substituted amino acid, as described herein. Accordingly, the percentage of “sequence similarity” is the percentage of amino acids which is either identical or conservatively changed; e.g., “sequence similarity” = (% sequence identity)+(% conservative changes). It should be understood that a sequence that has a specified percent similarity to a reference sequence necessarily encompasses a sequence with the same specified percent identity to that reference sequence. The skilled person will be aware of various computer programs, using different mathematical algorithms, that are available to determine the identity or similarity between two sequences. For instance, use can be made of a computer program employing the Needleman and Wunsch algorithm (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego U.S.A.); the algorithm of E. Meyers and W. Miller (Meyers et al. (1989)) which has been incorporated into the ALIGN program (version 2.0); or more preferably the BLAST (Basic Local Alignment Tool using default parameters); see e.g., US Patent 10,023,890, the content of which is incorporated by reference herein in its entirety.

[0173] As used herein, the phrase “maintains the same function”, when used in reference to an agonist or antagonist, means binding to the same receptor. As used herein, the phrase “maintains the same function”, when used in reference to an enzyme, means catalyzing the same reaction as a reference enzyme.

[0174] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. A wide variety of, site-specific mutagenesis approaches, e.g., Kunkel’s method, cassette mutagenesis, PCR site-directed mutagenesis (e.g., traditional PCR, primer extension, or inverse PCR), whole plasmid mutagenesis, in vivo site-directed mutagenesis, CRISPR / Cas-guided mutagenesis, are known in the art and can be applied by the ordinarily skilled artisan to introduce mutations into specific nucleic acid loci. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Braman, Jeff, ed. (2002) In Vitro Mutagenesis Protocols, Methods in Molecular Biology, Vol. 182 (2nd ed.); Khudyakov and Fields (2002), Artificial DNA: Methods and Applications, CRC Press; Hsu et al. (2014), Cell 157 (6): 1262-78; Cerchione et al. (2020) PLOS ONE 15 (4): e0231716; and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0175] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derivedfrom any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., plasmid DNA. Suitable RNA can include, e.g., mRNA.

[0176] The term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (e.g., mRNA) or antisense RNA derived from a nucleic acid fragment or fragments and / or to the translation of mRNA into a polypeptide.

[0177] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. As used herein, the term “detecting” or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.

[0178] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a cell, such as a probiotic microbe, is considered to be “engineered” when an exogenous nucleic acid is present in a vector within the microbe or introduced into the genome of the microbe. As another example, a polypeptide is considered to be “engineered” when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0179] In some embodiments of any of the aspects, the enteroendocrine modulating polypeptide described herein is exogenous. In some embodiments of any of the aspects, the enteroendocrine modulating polypeptide described herein is ectopic. In some embodiments of any of the aspects, the enteroendocrine modulating polypeptide described herein is not endogenous.

[0180] The term “exogenous” refers to a substance present in a cell other than its native source. The term “exogenous” when used herein can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term “endogenous” refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and / or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and / or at a different time. Ectopic also includes a substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.

[0181] In some embodiments of any of the aspects, the probiotic microbe comprises at least one functional heterologous gene. As used herein, the term “heterologous” refers to that which is not endogenous to, or naturally occurring in, a referenced sequence, molecule (including e.g., a protein), virus, cell, tissue, or organism. For example, a heterologous sequence of the present disclosure can be derived from a different species, or from the same species but substantially modified from an original form. Also for example, a nucleic acid sequence that is not normally expressed in a cell or a virus is a heterologous nucleic acid sequence with regard to that cell or virus. The term “heterologous” can refer to DNA, RNA, or protein that does not occur naturally as part of the organism in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature. It is DNA, RNA, or protein that is not endogenous to the virus or cell and has been artificially introduced into the virus or cell.

[0182] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g., a metabolic disease. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a metabolic disease. Treatment is generally “effective” if one or more symptoms or clinical markers arereduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0183] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g., a carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in or within nature.

[0184] As used herein, the term “administering,” refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an act of ingestion, injection, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0185] As used herein, the term “specific binding” refers to a chemical or physical interaction between two molecules, compounds, cells and / or particles (e.g., a receptor and an agonist or antagonist) wherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third non-target entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.

[0186] The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0187] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0188] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0189] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0190] As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0191] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[0192] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from,a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0193] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in cell biology, immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway’s Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin’s Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0194] Other terms are defined herein within the description of the various aspects of the invention.

[0195] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0196] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0197] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0198] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:1. A probiotic microbe engineered to secrete an enteroendocrine modulating polypeptide.2. The probiotic microbe of paragraph 1, which is a yeast.3. The probiotic microbe of paragraph 2, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae (5. cerevisiae), Saccharomyces boulardii (S. boulardii), and Saccharomyces unisporus (S. unisporus).4. The probiotic microbe of paragraph 1, which is a bacterium.5. The probiotic microbe of paragraph 4, wherein the bacterium belongs to a genus selected from the group consisting of Lactobacillus, Pediococcus, Streptococcus, Bacillus,Enter occous, Escherichia, Vibrio, Bacteroides, and Bifidobacterium .6. The probiotic microbe of any one of paragraphs 1-5, wherein the enteroendocrine modulating polypeptide comprises an enteroendocrine receptor agonist.7. The probiotic microbe of any one of paragraphs 1-5, wherein the enteroendocrine modulating polypeptide comprises an enteroendocrine receptor antagonist.8. The probiotic microbe of paragraph 6 or 7, wherein the enteroendocrine receptor is selected from the group consisting of taste receptor type 1 member 1 (TAS1R1), taste receptor type 1 member 2 (TAS1R2), taste receptor type 1 member 3 (TAS1R3), G Protein- Coupled Receptor 93 (GPR93), G-protein-couple receptor 6A (GPCR6A), G-protein-coupled receptor 142 (GPR142), calcium-sensing receptor (CaSR), Metabotropic Glutamate Receptor 1 (mGluRl), Metabotropic Glutamate Receptor 4 (mGluR4), PEPT1, and cholecystokinin 1 receptor (CCK-1R).9. The probiotic microbe of any one of paragraphs 1-5, wherein the enteroendocrine modulating polypeptide comprises a taste receptor type 1 member 3 (TAS1 / R3) receptor agonist and / or a taste receptor type 1 member 2 (TAS1 / R2) receptor agonist.10. The probiotic microbe of paragraph 9, wherein the TAS1 / R3 receptor and / or TAS1 / R2 agonist comprises a sweet protein.11. The probiotic microbe of paragraph 10, wherein the sweet protein comprises a singlechain polypeptide version of monellin.12. The probiotic microbe of paragraph 11, wherein the single-chain version of monellin comprises MNEI.13. The probiotic microbe of any one of paragraphs 1-12, wherein the enteroendocrine modulating polypeptide comprises a hormone selected from the group consisting ofcholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), glucagon- like peptide 2 (GLP-2), glicentin, oxyntomodulin, Peptide YY (PYY), secretin, Substance P (SP), xenin, glucosedependent insulinotropic polypeptide (GIP), neurotensin, motilin, Gcg (GLP1 precursor), ghrelin, somatostatin, gastrin, oxyntomodulin (OXM), and b-casomorphin.14. The probiotic microbe of any one of paragraphs 1-13, wherein the enteroendocrine modulating polypeptide comprises a peptide antagonist of Neuropeptide Y Receptor Y 1 (NpylR) or a peptide antagonist of dipeptidyl peptidase IV (DPP -IV).15. The probiotic microbe of any one of paragraphs 1-14, wherein the nucleic acid sequence encoding the enteroendocrine modulating polypeptide is codon-optimized for expression in the host probiotic microbe.16. A nucleic acid encoding and capable of expressing or directing the expression of an enteroendocrine modulating polypeptide, wherein the enteroendocrine modulating polypeptide is codon-optimized for expression in a yeast, and / or the enteroendocrine modulating polypeptide is operably linked to a yeast regulatory element.17. A nucleic acid encoding and capable of expressing an enteroendocrine modulating polypeptide, wherein the enteroendocrine modulating polypeptide is codon-optimized for expression in a bacterium, and / or the enteroendocrine modulating polypeptide is operably linked to a bacterial regulatory element.18. A vector comprising the nucleic acid of paragraph 16 or 17.19. A probiotic microbe comprising the nucleic acid of paragraph 16 or 17 or the vector of paragraph 18.20. A composition comprising the probiotic microbe of any one of paragraphs 1-15 or 19, or the nucleic acid of paragraph 16 or 17, or the vector of paragraph 18.21. The composition of paragraph 20, wherein the probiotic microbe is dried and viable.22. The composition of paragraph 20 or 21, wherein the probiotic microbe secretes the enteroendocrine modulating polypeptide in the stomach, duodenumjejunum, ileum, cecum, mid-colon, and / or distal colon of a subject.23. The composition of any one of paragraphs 20-22, wherein the probiotic microbe secretes the enteroendocrine modulating polypeptide in the distal colon of a subject.24. The composition of any one of paragraphs 20-23, wherein the probiotic microbe is formulated for delivery to the stomach, duodenum ejunum, ileum, cecum, mid-colon, and / or distal colon of a subject.25. The composition of any one of paragraphs 20-24, wherein the probiotic microbe is formulated for delivery to the distal colon of a subject.26. A pharmaceutical composition comprising the probiotic microbe of any one of paragraphs 1-15 or 19, or the nucleic acid of paragraph 16 or 17, or the vector of paragraph18, in combination with a pharmaceutically acceptable carrier.27. The pharmaceutical composition of paragraph 26, wherein the microbe is in spore or dried viable form.28. The pharmaceutical composition of any one of paragraphs 26-27, wherein the probiotic microbe is encapsulated.29. The pharmaceutical composition of paragraph 28, wherein the encapsulation comprises an enteric coating.30. Afood composition comprising the probiotic microbe of any one of paragraphs 1-15 or 19 or the composition of any one of paragraphs 20-25.31. The food composition of paragraph 30, further comprising a prebiotic that supports growth or colonization by the probiotic microbe.32. The food composition of paragraph 31, wherein the prebiotic is selected from amino acid (e.g., arginine, glutarate, and ornithine), biotin, fructooligosaccharide, galactooligosaccharide, hemi cellulose (e.g., arabinoxylan, xylan, xyloglucan, glucomannan), inulin, chitin, lactulose, mannan oligosaccharide, oligofructose-enriched inulin, gum (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectin (e.g., xylogal actouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soy fiber, sugarbeet fiber, pea fiber, com bran, and oat fiber), xylooligosaccharide, and polyamine (e.g., spermidine, putrescine).33. A medical food comprising the probiotic microbe of any one of paragraphs 1-15 or19.34. A supplement comprising the probiotic microbe of any one of paragraphs 1-15 or 19.35. A method of enteroendocrine modulation, the method comprising administering an enteroendocrine-modulating amount of the probiotic microbe of any one of paragraphs 1-15 or 19, the composition of any one of paragraphs 20-25, the pharmaceutical composition of any one of paragraphs 26-29, the food composition of any one of paragraphs 30-32, the medical food of paragraph 33, or the supplement of paragraph 34 to a subject in need of enteroendocrine modulation.36. The method of paragraph 35, wherein the enteroendocrine modulating polypeptide modulates metabolic energy homeostasis in the subject.37. The method of paragraph 36, wherein metabolic energy homeostasis modulation comprises modulation of glucose levels, modulation of metabolism, modulation of type 2 diabetes, weight management, satiety, gastric emptying, gut motility, nutrient absorption, gut hormone signaling, and / or management of dyslipidemia.38. The method of any one of paragraphs 35-37, wherein the probiotic microbe or the composition is administered daily.39. A method of modulating blood glucose homeostasis, the method comprising administering an enteroendocrine-modulating amount of a probiotic microbe engineered to secrete a polypeptide agonist of the T1R2-T1R3 sweet receptor or to secrete a polypeptide agonist of the GLP-1 receptor.40. A method of treating type II diabetes, the method comprising administering an enteroendocrine-modulating amount of a probiotic microbe engineered to secrete an agonist of the GLP-1 receptor.

[0199] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1

[0200] The ELITE platform described herein is a synthetic biology-based technology resource that permits the creation of safe and effective engineered live biotherapeutics (eLBPs). The platform provides a suite of technologies that are specifically designed for applications in human health and are effective for use in humans. The ELITE platform was used to create eLBPs that locally secrete genetically encoded molecular stimulators (GEMS) as agonists for the endogenous stimulation of gut hormones from enteroendocrine cells. The exemplified GEMS are sweet proteins; it should be understood that other enteroendocrine modulators can be produced and delivered in an analogous manner. Sweet proteins are naturally occurring plant-derived products that bind the taste receptor TAS1 / R3 to elicit a sweet perception that is 100 to 1,000 times greater than common ligands such as glucose (see e.g., Bilal M, 2022, Bioengineered. 13(4): 9815-9828). It has been described thatenteroendocrine cells in the distal intestine express TAS1 / R3 receptors and that their stimulation can contribute to the incretin response, regulating systemic glucose metabolism (see e.g., Ohtsu Y, 2014, Mol Cell Endocrinol. 394(1-2): 70-9). Ingestion of sugars and dietary components that can stimulate the distal colon sweet taste receptors are quickly assimilated or digested in the proximal small intestine, and therefore traditional oral delivery of these ligands cannot contribute to enteroendocrine stimulation in the large intestine. Targeted delivery to distal colon locations of effectors of TAS1 / R3 such as sweet proteins using the eLBP platform represents a strategy for the stimulation of enteroendocrine cells with therapeutic applications.

[0201] The engineered microbes can serve as a delivery vehicle for protein effectors to the entire length of the murine gastrointestinal tract. For that purpose, a strain of the yeast Saccharomyces boulardii was constructed that constitutively secretes the NANOLUC luminescent reporter enzyme, which allows the detection of the protein in complex samples using luminescence as a readout. An episomal vector was used that carries the origin of replication from the 2-micron circle plasmid and uses the aminoglycoside G418 as selectable marker. The vector harbors an expression cassette driven by the tdh3 (GDP) promoter and uses a secretion signal derived from the alpha mating factor. This episomal expression system was named pRG.

[0202] To investigate the protein delivery capacity into a mammalian gastrointestinal tract, 108CFUs of the NANOLUC-expressing S. boulardii (ELITE-011) was orally dosed into 8-week-old C57BL / 6J mice. Intestinal segments and fecal samples were collected 2 hours, 5 hours and 23 hours after the dosing of the Sbl 1 strain, from which ELITE-011 cell counts and luminesce readouts were obtained. During its transit through every segment of the GI tract, Sbl 1 cells were metabolically active, and they secreted detectable levels of the NANOLUC enzyme into the extracellular environment, with the highest signal happening in the distal colon at 5 hours post dosing. After 23 hours post dosing, the luciferase signal and cells counts were reduced more than three orders of magnitude, indicating that the vast majority Sbl 1 cells leave the murine guts as part of the normal intestinal transit and the residual fraction remain metabolically active and viable (see e.g., Fig. 1).

[0203] Having demonstrated that the system is functional for the delivery of protein effectors to the mammalian gut, a strain of the yeast Saccharomyces boulardii was next constructed that actively secretes the model sweet protein MNEI, which is a single chain polypeptide version of the naturally found sweet protein Monellin. The pRG expressionsystem was used, replacing the NANOLUC coding sequence with the yeast-codon optimized coding region of the MNEI gene. Upon growth under selective conditions in YTG media, the presence of the MNEI protein was confirmed in the supernatant of the cultures by SDS page (see e.g., Fig. 2).

[0204] A diet-induced obesity (DIO) mouse model was used to test the physiological effect of the delivery of an agonist of the TAS1 / R3 receptor, such as MNEI, throughout the mouse gastrointestinal tract. In the DIO mouse model, prolonged consumption of a high-fat diet results in metabolic dysregulation that leads to obesity and poor glycemic control. The metabolic effects on DIO mice were studied of a 15 -day treatment of daily oral dosing of 108CFU of the MNEI-secreting S. boulardii strain (ELITE-031) or a control S. boulardii strain (ELITE-029). As control treatments, a 15-day treatment of daily dosing of an equal volume of the delivery vehicle (Phosphate Buffer Saline, PBS) was performed on Normal Diet and DIO mice to obtain the metabolic parameters of a healthy and a diseased mice population, respectively (see e.g., Fig. 3A). After the treatment period, blood samples were collected, and relevant metabolic markers, adipokines and gut hormones were quantified using a custom- made U-PLEX MESOSCALE DISCOVERY ELISA plate (see e g., Fig. 3B). Upon z-score normalization of the metabolite data, t-distributed stochastic neighbor embedding (tSNE) was applied to represent the metabolic profiles in each treatment group in a two-dimensional matrix. DIO mice and normal diet mice treated with PBS formed two distinctive clusters representing a healthy and diseased metabolic profile (see e.g., Fig. 3C). While 50% of the mice that were treated with the control strain ELITE-029 belonged to the diseased state metabolic profile cluster, 88.8% of the mice treated with ELITE-031 were outside of this cluster. These results indicate that the treatment with the engineered S. boulardii that secretes MNEI can modify the enteroendocrine metabolite profile in DIO mice to a status that does not resemble the metabolic dysregulation caused by the continuous high-fat diet.

[0205] The DIO mice display a metabolic disease that resembles type 2 diabetes with elevated glycemia and poor glucose control. One of the principal effects of the stimulation of enteroendocrine hormones is the restoration of the glucose homeostasis in mice under a high- fat diet. The therapeutic effects on DIO mice glycemia after the treatment with ELITE-031 or ELITE-029 were determined. To obtain the glycemic parameters of a healthy and a metabolically disrupted mice population, the control treatments with PBS in the normal diet and DIO mice were used as references to bound the lower and upper limits of glycemia. After the 15-day treatment period, the basal glucose levels of DIO mice treated with eitherPBS or the ELITE-029 control strain were significantly higher than the normal diet mice. In contrast, the basal glycemia of DIO mice treated with ELITE-301 displayed lower concentrations of basal glucose that were not significantly different from the normal diet mice (see e.g., Fig. 4A). Furthermore, effects of the treatments in the metabolic capacity for regulating glycemia were investigated in an oral glucose tolerance test (OGTT) using a bolus of 2mg / kg in 6 hour fasted mice (see e.g., Fig. 4B). The glycemic excursion over a period of 90 minutes in the mice that received the ELITE-031 treatment were not significantly different from the glucose curve observed for the mice under normal diet conditions (see e.g., Fig. 4C). In contrast, DIO mice that received either the control strain ELITE-029 or PBS displayed significantly different glycemic excursions during the OGTT. These results indicate that the sustained delivery of MNEI to the gastrointestinal tract of DIO mice using an engineered strain promotes the reduction of glycemia that is not achievable by the treatment with a control S. boulardii strain and therefore represents an effective approach to modulate the enteroendocrine system to promote metabolic health.

[0206] Stimulation of the production of enteroendocrine hormones has been proposed as a potential therapeutic approach to address metabolic diseases. Pharmacologic approaches that use small molecules to trigger receptors involved in the release of gut hormones in enteroendocrine cells has proven unsuccessful due to off-target effects that results in unacceptable adverse events in patients (e.g., TAKEDA’s Fasiglifam / TAK-875). Oral delivery of natural or synthetic molecules that seek to stimulate endocrine hormones in distal locations of the intestine is usually ineffective due to the harsh physiological conditions of the GI tract that result in poor pharmacokinetics. The use of engineered gut-targeted microbes for the sustained delivery of molecular stimulators of enteroendocrine cells represents an effective approach to restore the molecular signals that are lost under microbial and metabolic dysregulation. The disclosed eLBPs act as delivery vehicles of molecules capable of modulating the enteroendocrine system in a controlled way that only a genetically engineered microorganism can afford.

[0207] Endogenous eLBP-mediated stimulation of gut hormones results in the activation of both endocrine and neuronal routes and increases efficacy of metabolic disease treatment, reduces risks and side effects, as well as offers the convenience of oral administration. The manufacturing of eLBPs under GMP conditions is highly scalable and therefore offers a costeffectiveness advantage for therapeutic interventions aimed at addressing large patients populations such as people living with metabolic syndrome.

Claims

CLAIMSWhat is claimed herein is:

1. A probiotic microbe engineered to secrete an enteroendocrine modulating polypeptide.

2. The probiotic microbe of claim 1, which is a yeast.

3. The probiotic microbe of claim 2, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae (5. cerevisiae), Saccharomyces boulardii (S. boulardii), and Saccharomyces unisporus (S. unisporus).

4. The probiotic microbe of claim 1, which is a bacterium.

5. The probiotic microbe of claim 4, wherein the bacterium belongs to a genus selected from the group consisting of Lactobacillus, Pediococcus, Streptococcus, Bacillus, Enter occous, Escherichia, Vibrio, Bacteroides, and Bifidobacterium .

6. The probiotic microbe of any one of claims 1-5, wherein the enteroendocrine modulating polypeptide comprises an enteroendocrine receptor agonist.

7. The probiotic microbe of any one of claims 1-5, wherein the enteroendocrine modulating polypeptide comprises an enteroendocrine receptor antagonist.

8. The probiotic microbe of claim 6 or 7, wherein the enteroendocrine receptor is selected from the group consisting of taste receptor type 1 member 1 (TAS1R1), taste receptor type 1 member 2 (TAS1R2), taste receptor type 1 member 3 (TAS1R3), G Protein-Coupled Receptor 93 (GPR93), G-protein-couple receptor 6A (GPCR6A), G-protein-coupled receptor 142 (GPR142), calcium-sensing receptor (CaSR), Metabotropic Glutamate Receptor 1 (mGluRl), Metabotropic Glutamate Receptor 4 (mGluR4), PEPT1, and cholecystokinin 1 receptor (CCK-1R).

9. The probiotic microbe of any one of claims 1-5, wherein the enteroendocrine modulating polypeptide comprises a taste receptor type 1 member 3 (TAS1 / R3) receptor agonist and / or a taste receptor type 1 member 2 (TAS1 / R2) receptor agonist.

10. The probiotic microbe of claim 9, wherein the TAS1 / R3 receptor and / or TAS1 / R2 agonist comprises a sweet protein.

11. The probiotic microbe of claim 10, wherein the sweet protein comprises a single-chain polypeptide version of monellin.

12. The probiotic microbe of claim 11, wherein the single-chain version of monellin comprises MNEI.

13. The probiotic microbe of any one of claims 1-12, wherein the enteroendocrine modulating polypeptide comprises a hormone selected from the group consisting of cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), glucagon- like peptide 2 (GLP-2), glicentin, oxyntomodulin, Peptide YY (PYY), secretin, Substance P (SP), xenin, glucosedependent insulinotropic polypeptide (GIP), neurotensin, motilin, Gcg (GLP1 precursor), ghrelin, somatostatin, gastrin, oxyntomodulin (OXM), and b-casomorphin.

14. The probiotic microbe of any one of claims 1-13, wherein the enteroendocrine modulating polypeptide comprises a peptide antagonist of Neuropeptide Y Receptor Y 1 (NpylR) or a peptide antagonist of dipeptidyl peptidase IV (DPP -IV).

15. The probiotic microbe of any one of claims 1-14, wherein the nucleic acid sequence encoding the enteroendocrine modulating polypeptide is codon-optimized for expression in the host probiotic microbe.

16. A nucleic acid encoding and capable of expressing or directing the expression of an enteroendocrine modulating polypeptide, wherein the enteroendocrine modulating polypeptide is codon-optimized for expression in a yeast, and / or the enteroendocrine modulating polypeptide is operably linked to a yeast regulatory element.

17. A nucleic acid encoding and capable of expressing an enteroendocrine modulating polypeptide, wherein the enteroendocrine modulating polypeptide is codon-optimized for expression in a bacterium, and / or the enteroendocrine modulating polypeptide is operably linked to a bacterial regulatory element.

18. A vector comprising the nucleic acid of claim 16 or 17.

19. A probiotic microbe comprising the nucleic acid of claim 16 or 17 or the vector of claim 18.

20. A composition comprising the probiotic microbe of any one of claims 1-15 or 19, or the nucleic acid of claim 16 or 17, or the vector of claim 18.

21. The composition of claim 20, wherein the probiotic microbe is dried and viable.

22. The composition of claim 20 or 21, wherein the probiotic microbe secretes the enteroendocrine modulating polypeptide in the stomach, duodenumjejunum, ileum, cecum, mid-colon, and / or distal colon of a subject.

23. The composition of any one of claims 20-22, wherein the probiotic microbe secretes the enteroendocrine modulating polypeptide in the distal colon of a subject.

24. The composition of any one of claims 20-23, wherein the probiotic microbe is formulated for delivery to the stomach, duodenum ejunum, ileum, cecum, mid-colon, and / or distal colon of a subject.

25. The composition of any one of claims 20-24, wherein the probiotic microbe is formulated for delivery to the distal colon of a subject.

26. A pharmaceutical composition comprising the probiotic microbe of any one of claims 1- 15 or 19, or the nucleic acid of claim 16 or 17, or the vector of claim 18, in combination with a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26, wherein the microbe is in spore or dried viable form.

28. The pharmaceutical composition of any one of claims 26-27, wherein the probiotic microbe is encapsulated.

29. The pharmaceutical composition of claim 28, wherein the encapsulation comprises an enteric coating.

30. Afood composition comprising the probiotic microbe of any one of claims 1-15 or 19 or the composition of any one of claims 20-25.

31. The food composition of claim 30, further comprising a prebiotic that supports growth or colonization by the probiotic microbe.

32. The food composition of claim 31, wherein the prebiotic is selected from amino acid (e.g., arginine, glutarate, and ornithine), biotin, fructooligosaccharide, galactooligosaccharide, hemi cellulose (e.g., arabinoxylan, xylan, xyloglucan, glucomannan), inulin, chitin, lactulose, mannan oligosaccharide, oligofructose-enriched inulin, gum (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectin (e.g., xylogal actouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soy fiber, sugarbeet fiber, pea fiber, com bran, and oat fiber), xylooligosaccharide, and polyamine (e.g., spermidine, putrescine).

33. A medical food comprising the probiotic microbe of any one of claims 1-15 or 19.

34. A supplement comprising the probiotic microbe of any one of claims 1-15 or 19.

35. A method of enteroendocrine modulation, the method comprising administering an enteroendocrine-modulating amount of the probiotic microbe of any one of claims 1-15 or 19, the composition of any one of claims 20-25, the pharmaceutical composition of any one of claims 26-29, the food composition of any one of claims 30-32, the medical food of claim 33, or the supplement of claim 34 to a subject in need of enteroendocrine modulation.

36. The method of claim 35, wherein the enteroendocrine modulating polypeptide modulates metabolic energy homeostasis in the subject.

37. The method of claim 36, wherein metabolic energy homeostasis modulation comprises modulation of glucose levels, modulation of metabolism, modulation of type 2 diabetes, weight management, satiety, gastric emptying, gut motility, nutrient absorption, gut hormone signaling, and / or management of dyslipidemia.

38. The method of any one of claims 35-37, wherein the probiotic microbe or the composition is administered daily.

39. A method of modulating blood glucose homeostasis, the method comprising administering an enteroendocrine-modulating amount of a probiotic microbe engineered to secrete a polypeptide agonist of the T1R2-T1R3 sweet receptor or to secrete a polypeptide agonist of the GLP-1 receptor.

40. A method of treating type II diabetes, the method comprising administering an enteroendocrine-modulating amount of a probiotic microbe engineered to secrete an agonist of the GLP-1 receptor.

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