Biosynthesis of protocatechuic acid

Abstract

Aspects of the present disclosure relate to the use of engineered host cells capable of producing protocatechuic acid (PCA). Aspects of the present disclosure also relate to compositions comprising engineered host cells capable of producing protocatechuic acid and / or cell constituents thereof, and the use of such compositions for altering and / or maintaining biological activity in a subject.

Description

[0001] 4)

[0002] BIOSYNTHESIS OF PROTOCATECHUIC ACID

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 711,589, filed October 24, 2024, the contents of which are incorporated by reference herein in their entirety.

[0005] STATEMENT REGARDING THE SEQUENCE LISTING

[0006] The instant application contains an electronic sequence listing which has been submitted in xml (ST.26 format concurrently herewith, herein incorporated by reference in its entirety. Said xml copy named “VI 18077 1040WO.xml” is 66,015 bytes in size, and was created on October 24, 2025.

[0007] FIELD OF THE DISCLOSURE

[0008] The present disclosure relates to engineered host cells capable of producing protocatechuic acid (PCA). The present disclosure also relates to compositions comprising engineered host cells capable of producing protocatechuic acid and / or cell constituents thereof.

[0009] BACKGROUND OF THE DISCLOSURE

[0010] Protocatechuic acid (PCA) is a dihydroxybenzoic acid that has been reported to possess many pharmacological activities, including antioxidant activity, anti-inflammatory activity, antimicrobial activity, cardiovascular protective activity, and neuroprotective activity. In addition, PCA serves as a precursor for synthesizing chemical compounds such as vanillin, muconic acid, and adipic acid.

[0011] Chemical synthesis of PCA is laborious and often results in low yields. There is a need in the art for alternative, more efficient methods for producing PCA, and molecules for which PCA is a precursor.

[0012] Provided herein are compositions and methods for producing PCA, precursors thereof, and molecules for which PCA is a precursor in microbial culture. The compositions disclosed herein involve an engineered S. boulardii strain comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant thereof. In some instances, the engineered S. boulardii is capable of producing PCA. As such, the compositions and methods disclosed herein are useful for producing PCA in vitro in a microbial culture.

[0013] In several commercial industries, PCA is sought after due to its role as a precursor for many high-value chemical compounds, such as vanillin, muconic acid, and adipic acid. In the pharmacological industry, PCA is sought after due to its characteristics as an antioxidant, anti- 4)

[0014] inflammatory compound, antimicrobial compound, cardiovascular protective compound, and neuroprotective compound. Host cells described in the present disclosure, including S. boulardii, capable of producing PCA may be useful in the production of compositions comprising high-level, high-quality PCA, precursors thereof, and molecules for which PCA serves as an intermediate molecule. Such host cells are also useful for the treatment of one or more diseases or disorders. Any disease, disorder or ailment for which PCA may alleviate at least one symptom are envisaged. In some embodiments, the host cells are probiotic. In some embodiments, the disclosure relates to a composition comprising an engineered probiotic host cell, such as a probiotic dietary supplement. Also disclosed are postbiotic compositions comprising said engineered host cells and / or cell constituents thereof. Such compositions are effective at modulating at least one biological activity in a subject.

[0015] SUMMARY OF THE DISCLOSURE

[0016] Provided herein are host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1.

[0017] In some embodiments of the above host cells, the AroZ or variant of AroZ comprises the amino acid sequence of SEQ ID NO: 1.

[0018] In some embodiments of the above host cells, the host cell further comprises at least a second heterologous polynucleotide sequence encoding each of the following genes: arol, aro3, aro4, tkll, and tall. In some embodiments of the above host cells, the host cell comprises at least two copies of the following genes: arol and aro3.

[0019] In some embodiments of the above host cells, the one or more of the heterologous polynucleotides are codon-optimized.

[0020] In some embodiments of the above host cells, the host cell is a yeast cell. In some embodiments, the yeast cell is a Saccharomyces cerevisiae cell or a Saccharomyces cerevisiae var. boulardii cell.

[0021] In some embodiments of the above host cells, the host cell is a probiotic strain. In some embodiments, the probiotic strain is Bacillus, Bacillus coagulans, Bacillus subtilis, Bifidobacterium, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidus regularis, Bifidobacterium bifidum, 4)

[0022] Bifidobacterium breve. Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifantis, Enterococcus, Escherichia, Escherichia coll, Lacticaseibacillus, Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, Lactiplanti bacillus plantarum, Lactobacillus, Lactobacillus acidophilus, L. brevis, Lactobacillus casei rhamnosus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, L. fermentum, L. gasseri, L. paracesei, L. reuteri, Ligilactobacillus, Ligilactobacillus salivarius, limosilactobacillus, limosilactobacillus fermentum, Limosilactobacillus reuteri, Limosilactobacillus reuteri, Pediococcus, Saccharomyces, Saccharomyces cerevisiae var. boulardii, Streptococcus, or Streptococcus thermophilus. In some embodiments, the probiotic strain is Saccharomyces cerevisiae var. boulardii.

[0023] Also provided herein are Saccharomyces cerevisiae var. boulardii host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, wherein said host cell further comprises the following heterologous polynucleotides encoding codon- optimized variants: i) a second heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkll enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; and vii) an eighth heterologous polynucleotide encoding a Tall enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7.

[0024] Also provided herein are microbial compositions comprising at least a first host cell, and / or cell constituent(s) thereof, wherein said at least first host cell comprises the host cell of any one of claims 1-11.

[0025] Also provided herein are microbial compositions comprising a plurality of Saccharomyces cerevisiae var. boulardii (S. boulardii) and / or cell constituent(s) thereof,

[0026] wherein said S. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of SEQ ID NO: 1.

[0027] In some embodiments of the above microbial compositions, said plurality comprises about 104CFU / gram to about 1012CFU / gram or about 104CFU / ml to about 1012CFU / ml of 5. boulardii.

[0028] In some embodiments of the above microbial compositions, said microbial composition further comprises protocatechuic acid (PCA). In some embodiments of the above microbial compositions, said PCA is present at a concentration of at least 0.0001 mM (i.e., 0.1 pM), at least 0.0005 mM, 0.001 mM (i.e., 1 pM), at least 0.005 mM, at least 0.01 mM, at least 0.03 mM, at least 0.06 mM, at least 0.12 mM, at least 0.25 mM, at least 0.5 mM, at least 1 mM, at least 2 mM. In some embodiments of the above microbial compositions, said PCA is present at a concentration of between about 0.005 mM (i.e., 5 pM) to about 0.06 mM.

[0029] In some embodiments of the above microbial compositions, said microbial composition further comprises i) luminal contents of the gastrointestinal tract of a subject; ii) at least one intestinal epithelial cell; and / or iii) at least one skin epithelial cell.

[0030] Also provided herein are Saccharomyces cerevisiae var. boulardii (S. boulardii) -derived postbiotic compositions, said composition comprising a plurality of S. boulardii, and / or cell constituent(s) thereof, wherein said S. boulardii comprises a heterologous polynucleotide encoding a Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1, and wherein 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%, or at least 99% of said plurality is non-viable.

[0031] In some embodiments of the above postbiotic compositions, said plurality comprises no more than: i) 1000 CFU / gram, 100 CFU / gram, 10 CFU / gram, or 1 CFU / gram; and / or ii) 1000 CFU / ml, 100 CFU / ml, 10 CFU / ml, or 1 CFU / ml, of said. boulardii.

[0032] In some embodiments of the above postbiotic compositions, said postbiotic composition further comprises protocatechuic acid (PCA). In some embodiments of the above postbiotic compositions, said PCA is present at a concentration of at least 0.0001 mM (i.e., 0.1 pM), at least 0.0005 mM, 0.001 mM (i.e., 1 pM), at least 0.005 mM, at least 0.01 mM, at least 0.03 mM, at least 0.06 mM, at least 0.12 mM, at least 0.25 mM, at least 0.5 mM, at least 1 mM, at least 2 mM. In some embodiments of the above postbiotic compositions, said PCA is present at a concentration of between about 0.005 mM (i.e., 5 pM) to about 0.06 mM.

[0033] In some embodiments of the above postbiotic compositions, said postibiotic composition further comprises i) luminal contents of the gastrointestinal tract of a subject; ii) at least one intestinal epithelial cell; and / or iii) at least one skin epithelial cell.

[0034] In some embodiments of the above microbial compositions or postbiotic compositions, said cell constituent(s) comprises at least one postbiotic agent. In some embodiments of the above microbial compositions or postbiotic compositions, said at least one postbiotic agent comprises a lipid, a carbohydrate, a lipoprotein, a glycolipid, a glycoprotein, a metabolite, genetic material, or any combination thereof.

[0035] In some embodiments of the above microbial compositions or postbiotic compositions, said microbial composition or said postbiotic composition further comprises a prebiotic. In some embodiments of the above microbial compositions or postbiotic compositions, said composition is formulated as a capsule, gel, softgel, hydrogel, paste, tablet, gummy, spray, aerosol, lozenge, effervescent tablet, orodispersible film, powder, or liquid. In some embodiments of the above microbial compositions or postbiotic compositions, the formulation is a liquid formulation. In some embodiments of the above microbial compositions or postbiotic compositions, the formulation is a solid formulation.

[0036] In some embodiments of the above microbial compositions or postbiotic compositions, said microbial composition or said postbiotic composition further comprises a nutritional supplement.

[0037] In some embodiments of the above microbial compositions or postbiotic compositions, said at least first microorganism is: i) lyophilized; ii) pasteurized; and / or iii) freeze-dried.

[0038] Also provided herein are food or beverage products comprising any of the microbial composition or postbiotic compositions disclosed herein.

[0039] In some embodiments of the above food or beverage products, said product is a nutritional whole food, drink, sports drink, coffee, tea, water, mineral water, soda, carbonated beverage, oral hydration drink, mineral water, soup, replacement food, nutritional bar, nutritional consumable, gummy, tablet, confectionery, fermented or unfermented milk-based product, yogurt product, milkbased powder, enteral nutritional product, fermented or unfermented cereal-based product, chocolate, milk, yogurt, cheese, ice cream, baby and infant formula, cereal, animal feed, protein shake, protein powder, functional waterjuice blend, smoothie, ready-to-drink (RTD) beverage, non-dairy milk, nut or seed butter, energy drink, functional shot, baked good, granola bar, snack bar, alcoholic beverage, frozen food, frozen meal, condiment, pasta, noodles, pet food, or pet treat.

[0040] Also provided herein are methods of altering or maintaining biological activity in a subject in need thereof, said method comprising administering to said subject an effective amount of any of the microbial compositions, postbiotic compositions, or food or beverage products disclosed herein.

[0041] In some embodiments of the above methods, said altered or maintained biological activity comprises any one of the following: i) suppressing expression of at least one gene associated with inflammation in at least one cell present in said subject; ii) stimulating an epithelial tissue response in said subject; iii) reducing at least one symptom of a gastrointestinal or skin disorder in said subject; iv) increasing levels of protocatechuic acid (PCA) in at least one tissue in said subject; and / or v) improving well-being of said subject, compared to a control, wherein said control comprises a subject who is not administered an effective amount of said composition.

[0042] In some embodiments of the above methods, said at least one gene is iNOS and / or COX-2.

[0043] In some embodiments of the above methods, said effective amount of the microbial composition comprises about 105CFU / gram to about 1012CFU / gram or about 105CFU / ml to about 1012CFU / ml of said 5. boulardii.

[0044] In some embodiments of the above methods, said composition is administered to the gastrointestinal tract of the subject.

[0045] Also provided herein are methods of producing a Saccharomyces cerevisiae var. boulardii (S. bou / ardiij-denved postbiotic composition comprising a plurality of S. boulardii, and / or cell constituent(s) thereof, said method comprising inoculating at least one S. boulardii cell into a cell culture media, incubating said inoculated culture medium under conditions suitable for growth and proliferation of said S. boulardii, and isolating a cell-free cell supernatant from said culture medium, thereby producing said postbiotic composition, wherein said S. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1, and wherein at least 90%, at least 95%, or at least 99% of said plurality is non-viable.

[0046] In some embodiments of the above methods, said incubation involves a fermentation step. 4)

[0047] In some embodiments of the above methods, the number of viable 5. boulardii cells are reduced.

[0048] In some embodiments of the above methods, said postbiotic composition is: i) lyophilized; ii) pasteurized; and / or iii) freeze-dried.

[0049] In some embodiments of the above methods, said method further comprises a step of formulating a food or beverage product comprising said postbiotic composition.

[0050] Also provided herein are methods of producing protocatechuic acid (PCA) in a microbial culture, said method comprising inoculating at least one cell of an engineered Saccharomyces cerevisiae van boulardii (S. boulardii) strain capable of producing PCA in a culture media, and incubating said medium under conditions suitable for growth and proliferation of said S. boulardii, whereby PCA is secreted into said medium, thereby producing a composition comprising PCA.

[0051] In some embodiments of the above methods, said PCA is present at a concentration of at least 12g / L.

[0052] In some embodiments of the above methods, said method further involves converting PCA into a second molecule. In some embodiments of the above methods, PCA is a precursor for said second molecule. In some embodiments of the above methods, said second molecule comprises vanillin, muconic acid, and / or adipic acid. In some embodiments of the above methods, said method comprises purifying said PCA or said second molecule.

[0053] In some embodiments of the above methods, said S. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented in this disclosure. The accompanying drawings are not intended to be drawn to scale. The drawings are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings: 4)

[0056] FIG. 1 shows a comparison of PCA production by wildtype and engineered strains of Saccharomyces var. boulardii cultured in Simulator of Human Intestinal Microbial Ecosystem (SHIME) medium when inoculated at an initial cell density of 106cells per milliliter.

[0057] FIG. 2 shows a comparison of PCA production by wildtype and engineered strains of Saccharomyces var. boulardii cultured in SHIME medium after 24 and 48 hours when inoculated at an initial cell density of 106cells per milliliter. The genotypes of Strains 2 to 6 in Figures 2 and 3 are described in Table 2.

[0058] FIG. 3 shows a comparison of PCA production by wildtype and engineered strains of Saccharomyces var. boulardii cultured in SHIME medium after 24 and 48 hours when inoculated at an initial cell density of 107cells per milliliter.

[0059] FIG. 4 shows a biochemical pathway for the production of PCA.

[0060] FIG. 5 shows a comparison of PCA production in three isolates of engineered Saccharomyces var. boulardii strain 3 cultured in fermentation medium for 32 hours.

[0061] FIGS. 6A-6B show reduced proinflammatory gene expression in mammalian cells contacted with LPS following pretreatment with a Saccharomyces cerevisiae var. boulardii-derived postbiotic composition. Gene expression was quantified via RT-qPCR at 4 hours, 8 hours, and 24 hours following LPS treatment and is presented as fold-change relative to no LPS controls. FIG. 6A depicts fold change in iNOS gene expression in cells exposed to LPS with and without postbiotic composition pretreatment. FIG.6B depicts fold change in COX-2 gene expression in cells exposed to LPS with and without postbiotic composition pretreatment.

[0062] FIG. 7 shows increased restitution of wounded epithelial tissue in the presence of a Saccharomyces cerevisiae var. boulardii-derived postbiotic composition having various concentrations of protocatechuic acid (PCA).

[0063] DETAILED DESCRIPTION OF THE DISCLOSURE

[0064] The present disclosure now will be described more fully hereinafter. The disclosure may be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure will satisfy applicable legal requirements.

[0065] Definitions 4)

[0066] 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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0067] As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells. Further, the term “a plant” may include a plurality of plants.

[0068] As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and / or” and not the exclusive sense of “either / or.”

[0069] The term “about” or “approximately” usually means within 5%, or more preferably within 1%, of a given value or range. When used in reference to numerical ranges, cutoffs, or specific values means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of an assay, result or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 5%, whichever is larger.

[0070] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0071] A “culture media” (or “growth media”) for the purposes of this disclosure, is a substance that provides essential nutrients for the growth of microorganisms. Culture media is used as a source of energy for one or more microorganisms, such as yeast, to ferment a particular product. For instance, and without limitation, culture media may be used to promote the growth of one or more microorganisms (e.g., a plurality of microbial cells). Culture media may include a solid, liquid, or gel substance. In a non-limiting exemplary embodiment, culture media may include a nutrient broth having meat extracts and peptone water. Nutrient broths may further include glucose, yeast, polysorbates, and derivatives thereof. Culture media may further include synthetic media wherein the synthetic media is a defined medium having known chemicals and a lack of yeast, plant, or animal tissue.

[0072] As used herein, an “effective amount” refers to a quantity of a composition disclosed herein necessary to perform any of the methods and / or uses describe herein. For instance, an effective amount of the postbiotic composition can suppress expression of at least one gene associated with inflammation in at least one cell present in said subject; stimulate an epithelial tissue response in said 4)

[0073] subject; reduce at least one symptom of a gastrointestinal or skin disorder in said subject; and / or improve well-being of said subject, and / or improve well-being of a subject when compared to an appropriate control. Appropriate controls for the methods are described herein. An effective amount of the postbiotic composition can be determined based on the desired outcome. The quantity to be administered to a subject, both according to number of treatments and effective amount, depends on the subject to be treated, the state of the subject, the environmental conditions of the subject, and the result desired. Precise amounts of the postbiotic composition also depend on the judgment of the practitioner and can be unique to each individual.

[0074] “Fermentation”, for the purposes of this disclosure, is a breakdown of a substance through the use of microorganisms (e.g., a plurality of microbial cells). For instance, and without limitation, fermentation (also referred to in this disclosure as a “fermentation process” or “fermenting”) may include the breakdown of an organic substrate by or using microorganisms such as, but not limited to, bacteria or yeast. In some embodiments of the method disclosed herein, fermentation includes the use of yeast. In some embodiments, fermentation comprises an aerobic process, wherein the aerobic process comprises a process occurring in the presence of oxygen. In some embodiments, fermentation comprises an anaerobic process, wherein the anaerobic process includes a process occurring in the absence of oxygen. In some embodiments, fermentation may include the use of a substrate and one or more microorganisms to create a postbiotic composition comprising PCA.

[0075] “Fermentation medium,” for the purposes of this disclosure, is one or more substances that provide for the growth of the plurality of microbial cells and facilitate the fermentation process. The fermentation medium may include sources of carbon, nitrogen, inorganic salts, vitamins, growth stimulants such as culture media as disclosed herein, liquids such as water, buffers, dissolved oxygen, enzymes, and any other substances that may be required for proper fermentation and / or growth of microorganisms during fermentation.

[0076] The term “genetic material” refers to any nucleic acid or polynucleotide, or fragments thereof. The genetic material described herein can be any polynucleotide or fragment thereof, derived from the genome of a microorganism. The nucleic acid can be DNA and / or RNA. DNA can comprise nDNA or mtDNA. Any type of RNA is included. For instance, RNA can be mRNA, tRNA, rRNA, snRNA, snoRNA, ncRNA, nmRNA, sRNA, gRNA, pcRNA, crRNA, tracrRNA, dsRNA, exRNA, hc-siRNA, hnRNA, lincRNA, IncRNA, miRNA, mrpRNA, circRNA, pre-mRNA, tmRNA, tasiRNA, stRNA, or shRNA. In some instances, the genetic material is RNA. For instance, the genetic material can be a microRNA. 4)

[0077] The term “inoculation” as used herein refers to the introduction of a microorganism (or plurality of microbial cells) to a substrate or composition. This may include placing the substrate in the presence of one or more microorganisms. Inoculation may include placing the substrate within a liquid containing one or more microorganisms or placing the microorganisms within a composition containing a substrate. For example, and without limitation, inoculation may include placing a microbial cell, or plurality of microbial cells, into a liquid solution containing a substrate, such as, for example, an organic substrate that has been made suitable for fermentation.

[0078] As used herein, the term “postbiotic agent” refers to inanimate microorganisms and / or their components. The term “inanimate” when used in reference to microorganisms of a postbiotic agent means lifeless, rather than ‘inactive, as this latter term might suggest an inert material. ‘Inanimate’ simply captures the fact that live microorganisms were present but have now been killed, without implying a loss of function. A postbiotic agent can include non- viable agents capable of being produced by a microorganism such as a bacteria (e.g., probiotic bacteria or lactic acid bacteria) or yeast or byproducts thereof. A postbiotic agent can be any substance derived from a microorganism, including compounds secreted by live bacteria (e.g., metabolic byproducts) or released after bacterial lysis, such as metabolites, enzymes, peptides, teichoic acids, peptidoglycan- derived muropeptides, polysaccharides, cell surface proteins, or organic acids, as described in Salminen S, Collado MC, Endo A, Hill C, Lebeer S, Quigley EMM, Sanders ME, Shamir R, Swann JR, Szajewska H, Vinderola G. The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat Rev Gastroenterol Hepatol. 2021 Sep; 18(9): 649-667, the contents of which are incorporated herein in their entirety. Any postbiotic agent can be present in the postbiotic composition. Substances derived from a microorganism can include fragments or constituents of a microbial cell that is nonviable and intact, or ruptured and lysed. For example, the postbiotic may be one or more cell wall fragments, exopolysaccharides, membrane vesicles, or genetic material that is released from an intact microbial cell following cell lysis. The terms “postbiotic” and “postbiotic agent” are used interchangeably herein. Postbiotics possess different functional properties. These properties can positively affect the microbiota homeostasis and / or the metabolic and signaling pathways in an individual, thus affecting specific physiological, immunological, neuro-hormone, biological, regulatory, or metabolic reactions. It would be understood that the microbial compositions and postbiotic compositions disclosed herein can comprise at least one postbiotic agent. The postbiotic agent can be at least one cell constituent(s) of an engineered S. boulardii cell disclosed herein, e.g., a S. boulardii cell comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3- 4)

[0079] dehydroshikimate dehydratase (AroZ) or a variant thereof. The postbiotic can be a molecule secreted by the cell. For instance, the postbiotic agent may be PCA produced by a S. boulardii cell.

[0080] As used herein, the term “protocatechuic acid” or “PCA” refers to the phenolic compound of 3,4-dihydroxybenzoic acid, which is found in many food plants, such as olives, white grapes, the pharmacological properties of PCA have been extensively investigated and are understood in the art, and involve, for example, anti-oxidant, anti-inflammatory, anti-hyperglycemic, and anti-apoptotic activities, as described in Semaming Y. et al., Pharmacological properties of protocatechuic Acid and its potential roles as complementary medicine. Evid Based Complement Alternat Med.

[0081] 2015;2015:593902, the contents of which are incorporated herein by reference in their entirety. Several studies have further shown that PCA is a major metabolite of complex polyphenols, especially anthocyanins. In some instances, the engineered S. boulardii disclosed herein are capable of producing PCA. In some instances, the engineered S. boulardii produce a variant of PCA. PCA variants retain the biological activity of natural PCA. In some instances, PCA variants may be modified to enhance at least one biological activity of wild-type PCA (i.e., PCA found in nature).

[0082] As used herein, the term “pro-inflammatory signal” refers to any molecule that initiates an inflammatory response (e.g., expression of genes, or secretion of molecules, associated with a pro-inflammatory response) in at least one host cell. Pro-inflammatory molecules in a subject, such as a mammalian subject, are readily understood in the art. Any pro-inflammatory molecule is included as a pro- inflammatory signal for the purposes of the present disclosure. In some instances, a pro-inflammatory signal comprises a cytokine. In another instance, a pro-inflammatory signal comprises a genetic material, such as a genetic material from a pathogenic or foreign microorganism. It would be understood that a pro- inflammatory signal can come into contact with a host cell to trigger or activate a pro- inflammatory response in said cell. Such response, at a tissue level, can contribute to a pro-inflammatory environment in said tissue.

[0083] As used herein with respect to a parameter, the term “decreased” or “decreasing” or “decrease” or “reduced” or “reducing” or “reduce” or “lower” or “loss” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) negative change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “decreased”, “reduced”, and the like encompass both a partial reduction and a complete reduction compared to a control. Controls are disclosed herein in the context of the methods utilized. 4)

[0084] As used herein with respect to a parameter, the term “increased” or “increasing” or “increase” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, or more) positive change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “increased”, “increase”, and the like encompass both a partial reduction and a significant increase compared to a control.

[0085] As used herein, the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0086] A “microbe” will be understood to be a microorganism, i.e. a microscopic organism, which can be single celled or multicellular. Microorganisms are very diverse and include all the bacteria, archaea, protozoa, fungi, and algae, especially cells of plant pathogens and / or plant symbionts. Certain animals are also considered microbes, e.g. rotifers.

[0087] A “nutritional supplement”, for the purposes of this disclosure, is an edible substance intended to provide nutritional value to a subject when consumed.

[0088] Various embodiments of this disclosure may be presented in a range format. It should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this disclosure. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1-10 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 1 to 6, from 1 to 7, from 1 to 8, from 1 to 9, from 2 to 4, from 2 to 6, from 2 to 8, from 2 to 10, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. This applies regardless of the breadth of the range.

[0089] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number “and” a second indicate number and “ranging / ranges from” a first indicate number 4)

[0090] “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. The recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the endpoints of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values ≥ 0 and ≤2 if the variable is inherently continuous.

[0091] The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

[0092] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.

[0093] Overview of the Disclosure

[0094] The present disclosure provides, in some aspects, engineered host cells (i.e., microorganism cells) capable of producing protocatechuic acid (PCA). In some aspects, the engineered host cell is a yeast cell. In such instances, the yeast cell is a S. boulardii comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant thereof. The present disclosure also provides, in some aspects, compositions comprising such engineered host cells and / or cell constituent(s) thereof. Such compositions include probiotic composition and postbiotic compositions comprising said engineered S. boulardii and / or cell constituents thereof. The probiotic compositions (e.g., microbial compositions) and postbiotic compositions disclosed herein can further comprise PCA.

[0095] Also disclosed herein are methods of producing microbial compositions and postbiotic compositions, and methods of using the same for altering or maintaining at least one biological activity in a tissue of a subject. For instance, the microbial compositions and postbiotic compositions disclosed 4)

[0096] herein find use in modulating and maintaining health and well-being of a subject. In particular, the compositions are useful in suppressing an inflammatory response in host cells, such as epithelial cells.

[0097] Methods of use include methods of suppressing expression of at least one gene associated with inflammation in at least one cell present in said subject; stimulating an epithelial tissue response in said subject; reducing at least one symptom of a gastrointestinal or skin disorder in said subject; increasing levels of protocatechuic acid (PCA) in at least one tissue in said subject; and / or improving well-being of said subject, compared to a control, wherein said control comprises a subject who is not administered an effective amount of said composition.

[0098] Protocatechuic Acid

[0099] Protocatechuic acid (PCA), also referred to as 3,4-dihydroxybenzoic acid, is a dihydroxybenzoic acid, a type of phenolic acid, that has been reported to possess many pharmacological activities, including antioxidant activity, anti-inflammatory activity, antimicrobial activity, cardiovascular protective activity, and neuroprotective activity. In addition, PCA serves as a precursor for synthesizing chemical compounds such as vanillin, muconic acid, and adipic acid. PCA is the compound of Formula 1:

[0100]

[0101] Enzymes Useful for the Production of PCA

[0102] Aspects of the present disclosure relate, at least in part, to the expression or over-expression in a host cell of one or more heterologous polynucleotides encoding one or more enzymes, e.g., enzymes useful for the production of PCA in the host cell. In some embodiments, expression (e.g., heterologous expression) in a host cell of a heterologous polynucleotide encoding an enzyme relates to introduction into a host cell of a heterologous polynucleotide encoding an enzyme, wherein the enzyme is not native to the host cell. In some embodiments, over-expression in a host cell of a heterologous 4)

[0103] polynucleotide encoding an enzyme relates to increased level, activity, and / or expression of an enzyme which is native to the host cell. In some embodiments, one or more enzymes useful for the production of PCA are members of the shikimate pathway. In some embodiments, a non-limiting example of a biochemical pathway for the production of PCA is provided in Figure 4, which notes one or more enzymes useful for the production of PCA. In some embodiments, the one or more enzymes is AroZ or a variant of AroZ. In some embodiments, the one or more enzymes is AroZ from Aspergillus oryzae or a variant thereof. In some embodiments, the one or more enzymes are AroZ, Arol, Aro3, Aro4, Tkll, and Tall. In some embodiments, the one or more enzymes are AroZ, Arol, Aro3, Aro4, Tkll, and Tall, wherein at least one of AroZ, Arol, Aro3, Aro4, Tkll, and Tall is from Saccharomyces cerevisiae var. boulardii. In some embodiments, the one or more enzymes are AroZ, Arol, Aro3, Aro4, Tkll, and Tall, and optionally one or more of: AroB, AroD and / or AroF. In some embodiments, one or more of the enzymes AroZ, Arol, Aro3, Aro4, Tkll, and Tall is from a GRAS (e.g., generally regarded as safe) organism. In some embodiments, each of the enzymes AroZ, Arol, Aro3, Aro4, Tkll, Tall, AroB, AroD or AroF is from a GRAS organism. In some embodiments, each of the enzymes AroZ, Arol, Aro3, Aro4, Tkll, Tall, AroB, AroD or AroF is from a GRAS organism and the host cell is also a GRAS organism. In some embodiments, each of the enzymes Arol, Aro3, Aro4, Tkll, Tall, AroB, AroD or AroF is from a GRAS organism, wherein the GRAS organism is Saccharomyces cerevisiae var. boulardii or Aspergillus oryzae. In some embodiments, each of the enzymes AroZ, Arol, Aro3, Aro4, Tkll, Tall, AroB, AroD or AroF is from a GRAS organism, wherein the GRAS organism is Saccharomyces cerevisiae var. boulardii or Aspergillus oryzae, and the host cell is Saccharomyces cerevisiae var. boulardii. In some embodiments, AroZ is from Aspergillus oryzae,' and each of Arol, Aro3, Aro4, Tkll, and Tall is from Saccharomyces cerevisiae var. boulardii,' and each of AroB, AroD and AroF is from E. coli. In some embodiments, AroZ is from Aspergillus oryzae,' and each of Arol, Aro3, Aro4, Tkll, Tall, AroB, AroD and AroF is from Saccharomyces cerevisiae var. boulardii, and the host cell is Saccharomyces cerevisiae var. boulardii (e.g., each of Arol, Aro3, Aro4, Tkll, and Tall is over-expressed in Saccharomyces cerevisiae var. boulardii. In some embodiments, a host cell comprises one or more heterologous polynucleotides encoding: AroZ or a variant thereof from Aspergillus oryzae,' and each of: Arol or a variant thereof, Aro3 or a variant thereof, Aro4 or a variant thereof, Tkll or a variant thereof, Tall or a variant thereof, which are each from Saccharomyces cerevisiae var. boulardii,' and AroB or a variant thereof, AroD or a variant thereof and AroF or a variant thereof, which are each from E. coli. In some embodiments, a host cell comprises one or more heterologous polynucleotides encoding: AroZ or a variant thereof 4)

[0104] from Aspergillus oryzae. and each of: Arol or a variant thereof, Aro3 or a variant thereof, Aro4 or a variant thereof, Tkll or a variant thereof, Tall or a variant thereof, which are each from Saccharomyces cerevisiae var. boulardii,' AroB or a variant thereof, AroD or a variant thereof and AroF or a variant thereof, which are each from E. coli; and the host cell is Saccharomyces cerevisiae var. boulardii. In some embodiments, the enzyme is dehydroshikimic acid dehydratase (AroZ). In some embodiments, the enzyme is AroZ from Aspergillus oryzae. In some embodiments, the enzyme AroZ is from Podospora. In some embodiments, the enzyme AroZ is from Neurospora. In some embodiments, the enzyme is Arol. In some embodiments, the enzyme is Arol from Saccharomyces cerevisiae var. boulardii. In some embodiments, the enzyme is Aro3. In some embodiments, the enzyme is Aro3 from Saccharomyces cerevisiae var. boulardii. In some embodiments, the enzyme is Aro4. In some embodiments, the enzyme is Aro4 from Saccharomyces cerevisiae var. boulardii. In some embodiments, the enzyme is AroB. In some embodiments, the enzyme is AroB from Escherichia coli. In some embodiments, the enzyme is AroD. In some embodiments, the enzyme is AroD from Escherichia coli. In some embodiments, the enzyme is AroF. In some embodiments, the enzyme is AroF from Escherichia coli. In some embodiments, an enzyme is transketolase 1 (Tkll). In some embodiments, the enzyme is Tkll from Saccharomyces cerevisiae var. boulardii. In some embodiments, the enzyme is Tall. In some embodiments, the enzyme is Tall from Saccharomyces cerevisiae var. boulardii.

[0105] In some embodiments, the enzyme is a variant enzyme. In some embodiments, the Arol is a variant Arol which has a mutation at position K1370, wherein the number indicates the position and the letter preceding the number indicates the amino acid at that position in a reference sequence (e.g., SEQ ID NO: 3). In some embodiments, the Aro3 is a variant Aro3 which has a mutation at position K222, wherein the number indicates the position and the letter preceding the number indicates the amino acid at that position in a reference sequence (e.g., SEQ ID NO: 4). In some embodiments, the Aro4 is a variant Aro4 which has a mutation at position K229, wherein the number indicates the position and the letter preceding the number indicates the amino acid at that position in a reference sequence (e.g., SEQ ID NO: 5). In some embodiments, a mutation is a substitution.

[0106] In some embodiments, the one or more enzymes described in the present disclosure is engineered. The term “engineered,” as used in the disclosure, refers to the product of modifying, mutating, or otherwise changing the native structure of a protein or enzyme. In some embodiments, one or more of the enzymes described in the present disclosure is an engineered enzyme. In some embodiments, an engineered enzyme comprises one or more amino acid additions, deletions, or 4)

[0107] substitutions relative to a wildtype or reference enzyme. In some embodiments, an engineered enzyme is encoded by an engineered polynucleotide. In some embodiments, an engineered polynucleotide comprises one or more nucleotide additions, deletions, or point mutations relative to a reference polynucleotide.

[0108] In some embodiments, an enzyme is an AroZ from Aspergillus oryzae or a variant thereof. In some embodiments, the AroZ from Aspergillus oryzae is ATOZAO. In some embodiments, the ATOZAO is codon-optimized. The amino acid sequence of ATOZAO is provided by SEQ ID NO: 1. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 1 is provided by SEQ ID NO: 14.

[0109] In some embodiments, an enzyme is an AroZ from Podospora or a variant thereof. In some embodiments, the AroZ from Podospora is AroZps. In some embodiments, the AroZps is codon-optimized. The amino acid sequence of AroZps is provided by SEQ ID NO: 2. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 2 is provided by SEQ ID NO: 15.

[0110] In some embodiments, an enzyme is an Arol from Saccharomyces cerevisiae var. boulardii or a variant thereof. In some embodiments, the Arol from Saccharomyces cerevisiae var. boulardii is Arol SB. In some embodiments, the Arol SB is codon-optimized. The amino acid sequence of Arol SB is provided by SEQ ID NO: 3. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 3 is provided by SEQ ID NO: 16.

[0111] In some embodiments, the Arol SB is a variant Arol SB. In some embodiments, the variant Arol SB comprises an amino acid substitution at position KI 370 relative to the sequence of SEQ ID NO: 3. In some embodiments, the variant Arol SB comprises a K1370A amino acid substitution relative to the sequence of SEQ ID NO: 3.

[0112] The amino acid sequence of a variant Arol SB comprising a K1370A amino acid substitution relative to SEQ ID NO: 3 is provided by SEQ ID NO: 11. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 11 is provided by SEQ ID NO: 24.

[0113] In some embodiments, an enzyme is an Aro3 from Saccharomyces cerevisiae var. boulardii or a variant thereof. In some embodiments, the Aro3 from Saccharomyces cerevisiae var. boulardii is Aro3sB. In some embodiments, the Aro3sB is codon-optimized. The amino acid sequence of Aro3sB is provided by SEQ ID NO: 4. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 4 is provided by SEQ ID NO: 17.

[0114] In some embodiments, the Aro3sB is a variant Aro3sB. In some embodiments, the variant Aro3sB comprises an amino acid substitution at position K222 relative to the sequence of SEQ ID NO: 4. In some embodiments, the variant Aro3sB comprises a K222L amino acid substitution relative to the 4)

[0115] sequence of SEQ ID NO: 4. The amino acid sequence of a variant Aro3sB comprising a K222L amino acid substitution relative to SEQ ID NO: 4 is provided by SEQ ID NO: 12. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 12 is provided by SEQ ID NO: 25.

[0116] In some embodiments, an enzyme is an Aro4 from Saccharomyces cerevisiae var. boulardii or a variant thereof. In some embodiments, the Aro4 from Saccharomyces cerevisiae var. boulardii is Aro4sB. In some embodiments, the Aro4sB is codon-optimized. The amino acid sequence of Aro4sB is provided by SEQ ID NO: 5. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 5 is provided by SEQ ID NO: 18.

[0117] In some embodiments, the Aro4sB is a variant Aro4sB. In some embodiments, the variant Aro4sB comprises an amino acid substitution at position K229 relative to the sequence of SEQ ID NO: 5. In some embodiments, the variant Aro4sB comprises a K229L amino acid substitution relative to the sequence of SEQ ID NO: 5. The amino acid sequence of a variant Aro4sB comprising a K229L amino acid substitution relative to SEQ ID NO: 5 is provided by SEQ ID NO: 13. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 13 is provided by SEQ ID NO: 26.

[0118] In some embodiments, an enzyme is an Tkll from Saccharomyces cerevisiae var. boulardii or a variant thereof. In some embodiments, the Tkll from Saccharomyces cerevisiae var. boulardii is Tkll SB. In some embodiments, the TkllsB is codon-optimized. The amino acid sequence of TkllsB is provided by SEQ ID NO: 6. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 6 is provided by SEQ ID NO: 19.

[0119] In some embodiments, an enzyme is an Tall from Saccharomyces cerevisiae var. boulardii or a variant thereof. In some embodiments, the Tall from Saccharomyces cerevisiae var. boulardii is Tall SB. In some embodiments, the TallsB is codon-optimized. The amino acid sequence of TallsB is provided by SEQ ID NO: 7. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 7 is provided by SEQ ID NO: 20.

[0120] In some embodiments, an enzyme is an AroB from Escherichia coli or a variant thereof. In some embodiments, the AroB from Escherichia coli is AroBEC. In some embodiments, the polynucleotide encoding the enzyme AroBEC is codon-optimized. The amino acid sequence of AroBEC is provided by SEQ ID NO: 8. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 8 is provided by SEQ ID NO: 21.

[0121] In some embodiments, an enzyme is an AroD from Escherichia coli or a variant thereof. In some embodiments, the AroD from Escherichia coli is AroDEC. In some embodiments, the AroDEC is 4)

[0122] codon- optimized. The amino acid sequence of AroDEC is provided by SEQ ID NO: 9. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 9 is provided by SEQ ID NO: 22.

[0123] In some embodiments, an enzyme is an AroF from Escherichia coli or a variant thereof. In some embodiments, the AroF from Escherichia coli is AroFEC. In some embodiments, the AroFEC is codon- optimized. The amino acid sequence of AroFEC is provided by SEQ ID NO: 10. A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 10 is provided by SEQ ID NO: 23.

[0124] Variants

[0125] Aspects of the disclosure relate to variant enzymes. As used in this disclosure, a "variant" polynucleotide refers to a polynucleotide that differs from a reference polynucleotide by one or more nucleotides in its sequence. For polynucleotides, a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and / or 3' end; deletion and / or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and / or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. Generally, variants of a particular polynucleotide of the invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein. As used in this disclosure, a "variant" polypeptide refers to a polypeptide that differs from a reference polypeptide by one or more amino acids in its sequence. A variant amino acid or protein is intended to mean an amino acid or protein derived from the native amino acid or protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and / or C-terminal end of the native protein; deletion and / or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein. Biologically active variants of a native polypeptide will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native sequence as determined by sequence alignment programs and parameters described herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. 4)

[0126] A variant may share at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity with a reference sequence, including all values in between.

[0127] Unless otherwise noted, the term “sequence identity” refers to the relatedness of the sequences of two polypeptides or polynucleotides when the sequences are aligned, and the term “percent identity” refers to the percentage of residues (amino acids or nucleotides) that are identical when two polypeptide or polynucleotide sequences are aligned. In some embodiments, sequence identity and / or percent identity is determined across the entire length of a sequence, while in other embodiments, sequence identity and / or percent identity is determined over a region of a sequence.

[0128] Percent identity of polypeptide or polynucleotide sequences can be calculated by any of the methods known to one of ordinary skill in the art. For example, percent identity can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST® and XBLAST® programs (version 2.0) of Altschul et al., J. Mol. Biol.

[0129] 215:403-10, 1990. BLAST® protein searches can be performed, for example, with the XBLAST program, score=50, wordlength=3. Where gaps exist between two sequences, Gapped BLAST® can be utilized, for example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST® and Gapped BLAST® programs, the default parameters of the respective programs (e.g., XBLAST® and NBLAST®) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.

[0130] A second example of a local alignment technique is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) J. Mol. Biol. 147:195-197). An example of a global alignment technique is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) J. Mol. Biol. 48:443-453), which is based on dynamic programming. A further example of a global alignment technique is the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA).

[0131] In some embodiments, the identity of two polypeptide sequences is determined by aligning the amino acid sequences of the polypeptides, calculating the number of identical amino acids, and dividing by the length of one of the polypeptide sequences. In some embodiments, the identity of two 4)

[0132] polynucleotide sequences is determined by aligning the nucleotide sequences of the polynucleotides, calculating the number of identical nucleotides and dividing by the length of one of the polynucleotide sequences.

[0133] For multiple sequence alignments, computer programs including Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct 11;7:539) may be used.

[0134] In some embodiments, sequence identity is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993 (e.g., BLAST®, NBLAST®, XBLAST® or Gapped BLAST® programs, using default parameters of the respective programs).

[0135] In some embodiments, the sequence identity of two amino acid or polynucleotide sequences is determined using the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) J. Mol. Biol.

[0136] 147: 195-197) or the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) J. Mol. Biol. 48:443-453).

[0137] In some embodiments, the sequence identity of two amino acid or polynucleotide sequences is determined using a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA).

[0138] In some embodiments, the sequence identity of two amino acid or polynucleotide sequences is determined using Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct 11;7:539).

[0139] As used in this application, a residue (such as a nucleic acid residue or an amino acid residue) in sequence “X” is referred to as corresponding to a position or residue (such as a nucleic acid residue or an amino acid residue) “Z” in a different sequence “Y” when the residue in sequence “X” is at the counterpart position of “Z” in sequence “Y” when sequences X and Y are aligned using sequence alignment tools known in the art.

[0140] Variant sequences may be homologous sequences. As used in this application, homologous sequences are sequences (e.g., nucleic acid or amino acid sequences) that share a certain percent identity (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 100% percent identity, including all values in between) and may be paralogous sequences, orthologous sequences, or sequences arising from convergent evolution. Paralogous sequences arise from duplication of a gene 4)

[0141] within a genome of a species, while orthologous sequences diverge after a speciation event. Two different species may have evolved independently but may each comprise a sequence that shares a certain percent identity with a sequence from the other species as a result of convergent evolution.

[0142] In some embodiments, a polypeptide variant comprises the same or substantially similar secondary structure (e.g., alpha helix, beta sheet) as that of a reference polypeptide. In some embodiments, a polypeptide variant comprises the same or substantially similar tertiary structure as that of a reference polypeptide. As a non-limiting example, a variant polypeptide may have low primary sequence identity (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% sequence identity) compared to a reference polypeptide, but comprises one or more secondary structures (e.g., including but not limited to loops, alpha helices, or beta sheets) or the same tertiary structure as that of a reference polypeptide. For example, a loop may be located between a beta sheet and an alpha helix, between two alpha helices, or between two beta sheets. Homology modeling may be used to compare two or more tertiary structures.

[0143] Mutations can be made in a nucleotide sequence using any method known to one of ordinary skill in the art. For example, mutations can be made using gene editing tools, PCR, site-directed mutagenesis (e.g., according to Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), chemical synthesis of a gene or polypeptide, or by insertions, such as insertion of a tag (e.g., a HIS tag or a GFP tag), as well as using chemical mutagens or ultraviolet light. Mutations can include, for example, one or more substitutions, deletions, additions, insertions, fusions, translocations, or any combination thereof, generated using any method known in the art.

[0144] In some embodiments, methods for producing variants include methods for generating circular permutation of a sequence (Yu and Lutz, Trends Biotechnol. 2011 Jan;29(1):18-25). To generate a circular permutation of a polypeptide sequence, the linear primary sequence of a polypeptide can be circularized (e.g., by joining the N-terminal and C-terminal ends of the sequence) and the resulting circular polypeptide can be re-linearized, i.e. is severed (“broken”) at a different location, to generate a linear polypeptide with different N and C termini, the resulting polypeptide being a circular permutation of the original sequence. Thus, the linear primary sequence of the new polypeptide may have low sequence identity (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less or less than 5%, including all values in 4)

[0145] between) compared to the linear sequence of the polypeptide before it was circularized and severed as determined by linear sequence alignment methods (e.g., Clustal Omega or BLAST). Topological analysis of the two polypeptides, however, may reveal that the tertiary structure of the two polypeptides is similar or dissimilar. Without being bound by a particular theory, a variant polypeptide created through circular permutation of a reference polypeptide and with a similar tertiary structure as the reference polypeptide can share similar functional characteristics (e.g., enzymatic activity, enzyme kinetics, substrate specificity or product specificity). In some instances, circular permutation may alter the secondary structure, tertiary structure or quaternary structure and produce a polypeptide with different functional characteristics (e.g., increased or decreased enzymatic activity, different substrate specificity, or different product specificity). See, e.g., Yu and Lutz, Trends Biotechnol. 2011 Jan;29(1):18-25.

[0146] It should be appreciated that in a polypeptide that has undergone circular permutation, the linear amino acid sequence of the polypeptide would differ from a reference polypeptide that has not undergone circular permutation. However, one of ordinary skill in the art would be able to determine which residues in the polypeptide that has undergone circular permutation correspond to residues in the reference polypeptide that has not undergone circular permutation by, for example, aligning the sequences and detecting conserved motifs, and / or by comparing the structures or predicted structures of the polypeptides, e.g., by homology modeling.

[0147] In some embodiments, an algorithm that determines the percent identity between a sequence of interest and a reference sequence described in this application accounts for the presence of circular permutation between the sequences. The presence of circular permutation may be detected using any method known in the art, including, for example, RASPODOM (Weiner et al., Bioinformatics. 2005 Apr l;21(7):932-7). In some embodiments, the presence of circulation permutation is corrected for (e.g., the domains in at least one sequence are rearranged) prior to calculation of the percent identity between a sequence of interest and a sequence described in this application.

[0148] Functional variants of enzymes disclosed in this application are also encompassed by the present disclosure. For example, functional variants may bind one or more of the same substrates or produce one or more of the same products (e.g., one or more precursors of PCA or a product for which PCA is a precursor). Functional variants may be identified using any method known in the art. For example, the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 described above may be used to identify homologous proteins. 4)

[0149] Putative functional variants may also be identified by searching for polypeptides with functionally annotated domains. Databases including Pfam (Sonnhammer et al., Proteins. 1997 Jul;28(3):405-20) may be used to identify polypeptides with a particular domain.

[0150] Homology modeling may also be used to identify amino acid residues that are amenable to mutation without affecting function. A non-limiting example of such a method may include use of position-specific scoring matrix (PSSM) and an energy minimization protocol. See, e.g., Stormo et al., Nucleic Acids Res. 1982 May ll;10(9):2997-3011.

[0151] PSSM may be paired with calculation of a Rosetta energy function, which determines the difference between the wild-type and a mutant, such as a point mutant. Without being bound by a particular theory, potentially stabilizing mutations can be desirable for protein engineering (e.g., production of functional homologs). In some embodiments, a potentially stabilizing mutation has a AAGca / cvalue of less than -0.1 (e.g., less than -0.2, less than -0.3, less than -0.35, less than -0.4, less than -0.45, less than -0.5, less than -0.55, less than -0.6, less than -0.65, less than -0.7, less than -0.75, less than -0.8, less than -0.85, less than -0.9, less than -0.95, or less than -1.0) Rosetta energy units (R.e.u.). See, e.g., Goldenzweig etal., Mol Cell. 2016 Jul 21;63(2):337-346. doi:

[0152] 10.1016 / j.molcel.2016.06.012.

[0153] In some embodiments, a polynucleotide sequence encoding an enzyme associated with the disclosure comprises a mutation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more than 100 nucleotide positions corresponding to a reference sequence. In some embodiments, the polynucleotide sequence encoding an enzyme associated with the disclosure comprises a mutation in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more codons of a coding sequence relative to a reference coding sequence. As will be understood by one of ordinary skill in the art, a mutation within a codon may or may not change the amino acid that is encoded by the codon due to degeneracy of the genetic code. In some embodiments, the one or more mutations in the coding sequence do not alter the amino acid sequence of the coding sequence relative to the amino acid sequence of a reference polypeptide. 4)

[0154] In some embodiments, the one or more mutations in a polynucleotide sequence encoding an enzyme alters the amino acid sequence of the polypeptide relative to the amino acid sequence of a reference polypeptide. In some embodiments, the one or more mutations alter the amino acid sequence of the polypeptide relative to the amino acid sequence of a reference polypeptide and alter (enhance or reduce) an activity of the polypeptide relative to the reference polypeptide.

[0155] The activity, including specific activity, of any of the polypeptides described in this application may be measured using methods known in the art. As a non-limiting example, a polypeptide’s activity may be determined by measuring its substrate specificity, product(s) produced, the concentration of product(s) produced, or any combination thereof. As used in this application, “specific activity” of a polypeptide refers to the amount (e.g., concentration) of a particular product produced for a given amount (e.g., concentration) of the polypeptide per unit time.

[0156] Mutations in a polypeptide coding sequence may result in conservative amino acid substitutions. As used in this application, a “conservative amino acid substitution,” or “conservatively substituted amino acid” refers to an amino acid substitution that does not alter the relative charge or size characteristics or functional activity of the protein in which the amino acid substitution is made.

[0157] In some instances, an amino acid is characterized by its R group (see, e.g, Table 1). For example, an amino acid may comprise a nonpolar aliphatic R group, a positively charged R group, a negatively charged R group, a nonpolar aromatic R group, or a polar uncharged R group. Non-limiting examples of an amino acid comprising a nonpolar aliphatic R group include alanine, glycine, valine, leucine, methionine, and isoleucine. Non-limiting examples of an amino acid comprising a positively charged R group include lysine, arginine, and histidine. Non-limiting examples of an amino acid comprising a negatively charged R group include aspartate and glutamate. Non-limiting examples of an amino acid comprising a nonpolar, aromatic R group include phenylalanine, tyrosine, and tryptophan. Non-limiting examples of an amino acid comprising a polar uncharged R group include serine, threonine, cysteine, proline, asparagine, and glutamine.

[0158] Functionally equivalent variants of polypeptides may include conservative amino acid substitutions. Non-limiting examples of conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Additional non-limiting examples of conservative amino acid substitutions are provided in Table 1. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 residues can be changed when preparing variant polypeptides. In some embodiments, amino acids are replaced by conservative amino acid substitutions.

[0159] Table 1. Non-limiting examples of conservative amino acid substitutions

[0160]

[0161] 4)

[0162]

[0163] Amino acid substitutions in the amino acid sequence of a polypeptide to produce a polypeptide variant having a desired property and / or activity can be made by alteration of the coding sequence of the polypeptide. Similarly, conservative amino acid substitutions in the amino acid sequence of a polypeptide to produce functionally equivalent variants of the polypeptide typically are made by alteration of the coding sequence of the polypeptide.

[0164] Polynucleotides Encoding Enzymes Useful for the Production PCA

[0165] Aspects of the present disclosure relate to recombinant enzymes, functional modifications and variants thereof, polynucleotides encoding said enzymes, as well as uses relating to any thereof. These enzymes include AroZ, Arol, Aro3, Aro4, Tkll, Tall, and variants thereof; and optionally: AroB, AroD, and AroF, and variants thereof. For example, the enzymes and cells described in this application may be used to produce increased titers of PCA relative to an enzyme that is not a variant or a cell that does not comprise a variant enzyme. The methods may comprise using a host cell comprising one or more enzymes disclosed in this application, a cell lysate, isolated enzymes, or any combination thereof. Methods comprising recombinant expression of polynucleotides encoding an enzyme disclosed in this application in a host cell are encompassed by the present disclosure. In vitro methods comprising reacting one or more enzymes described in the present disclosure in a reaction mixture disclosed in this application are also encompassed by the present disclosure.

[0166] The term “heterologous” with respect to a polynucleotide, such as a polynucleotide comprising a gene, is used interchangeably with the term “exogenous” and the term “recombinant” and refers to: a polynucleotide that has been artificially supplied to a biological system such as a cell; a polynucleotide that has been modified within a biological system; or a polynucleotide whose expression or regulation has been manipulated within a biological system. A heterologous polynucleotide that is introduced into or expressed in a host cell may be a synthetic polynucleotide, a polynucleotide that comes from a different organism or species from the host cell, or a polynucleotide that results from modification or selective editing within the host cell of a polynucleotide that is endogenous to the host cell. A polynucleotide comprising a sequence that is endogenous to a host cell also may be considered 4)

[0167] heterologous when it is, for example: situated non-naturally in the host cell; expressed recombinantly in the host cell, either stably or transiently; present in a copy number that differs from the naturally occurring copy number within the host cell; or expressed in a non-natural way or at a non-natural level within the host cell, such as through manipulation of regulatory regions that control expression of the polynucleotide. In some embodiments, a heterologous polynucleotide is a polynucleotide that comprises a sequence endogenous to a host cell but whose expression is driven by a promoter that does not naturally regulate expression of the polynucleotide. In other embodiments, a heterologous polynucleotide is a polynucleotide that comprises a sequence endogenous to the host cell and whose expression is driven by a promoter that does naturally regulate expression of the polynucleotide, but the promoter driving its expression or another regulatory region regulating its expression has been modified. In some embodiments, the promoter is recombinantly activated or repressed. For example, gene-editing techniques may be used to regulate expression of a polynucleotide in a cell, including an endogenous polynucleotide, from a promoter, including an endogenous promoter. See, e.g., Chavez et al., Nat Methods. 2016 Jul; 13(7): 563-567. A heterologous polynucleotide may comprise a wild-type sequence or a mutant sequence as compared with a reference polynucleotide sequence.

[0168] A polynucleotide encoding an enzyme associated with the disclosure, may be incorporated into any appropriate vector through any method known in the art. For example, the vector may be an expression vector, including but not limited to a viral vector (e.g., a lentiviral, retroviral, adenoviral, or adeno-associated viral vector), any vector suitable for transient expression, any vector suitable for constitutive expression, or any vector suitable for inducible expression (e.g., a galactose-inducible or doxycycline-inducible vector). The vector may be a cloning vector, such as a plasmid, fosmid, phagemid, virus genome or artificial chromosome.

[0169] As used in this application, the term "expression vector" or "expression construct" refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide in a host cell, such as a yeast cell or bacterial cell. In some embodiments, a polynucleotide associated with the disclosure is inserted into an expression vector or expression construct such that it is operably joined to regulatory sequences and, in some embodiments, expressed as an RNA transcript. In some embodiments, the expression vector or expression construct contains one or more markers, such as a selectable marker, to identify cells transformed or transfected with the expression vector or expression construct. A polynucleotide encoding a polypeptide associated with the disclosure is “operably joined” or “operably linked” to a regulatory sequence when the polynucleotide and the regulatory sequence are covalently linked, and 4)

[0170] the expression or transcription of the polynucleotide is under the influence or control of the regulatory sequence.

[0171] In some embodiments, a polynucleotide encoding any of the polypeptides described in this application is under the control of regulatory sequences (e.g., enhancer sequences). In some embodiments, a polynucleotide (e.g., a polynucleotide comprising a gene) is expressed under the control of a promoter. In some embodiments, the promoter is a native promoter, corresponding to the promoter of the gene in its endogenous context. In other embodiments, the promoter is not the native promoter of the gene, e.g., the promoter is different from the promoter of the gene in its endogenous context.

[0172] In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, GALI, GAL10, GAL7, GAL3, GAL2, MET3, MET25, HXT3, HXT7, ACT1, ADH1, ADH2, CUP1-1, ENO2, and SOD1, as would be known to one of ordinary skill in the art (see, e.g., Addgene website: blog.addgene.org / plasmids-101-the-promoter-region). In some embodiments, the promoter is a prokaryotic promoter (e.g., bacteriophage or bacterial promoter). Non-limiting examples of bacteriophage promoters include Pls Icon, T3, T7, SP6, and PL. Non-limiting examples of bacterial promoters include Pbad, PmgrB, Ptrc2, Plac / ara, Ptac, and Pm.

[0173] In some embodiments, the promoter is an inducible promoter. As used in this application, an “inducible promoter” is a promoter controlled by the presence or absence of a molecule. Non-limiting examples of inducible promoters include chemically regulated promoters and physically regulated promoters. For chemically regulated promoters, the transcriptional activity can be regulated by one or more compounds, such as alcohol, an antibiotic such as tetracycline, a carbon source such as galactose, a steroid, a metal, or other compounds. For physically regulated promoters, transcriptional activity can be regulated by a phenomenon such as light or temperature. Non-limiting examples of tetracycline-regulated promoters include anhydrotetracycline (aTc)-responsive promoters and other tetracyclineresponsive promoter systems (e.g., a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)). Non-limiting examples of steroid-regulated promoters include promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily. Non-limiting examples of metal-regulated promoters include promoters derived from metallothionein (proteins that bind and sequester metal ions) genes. Non-limiting examples of pathogenesis-regulated promoters include promoters induced by salicylic acid, ethylene or 4)

[0174] benzothiadiazole (BTH). Non-limiting examples of temperature / heat-inducible promoters include heat shock promoters. Non-limiting examples of light-regulated promoters include light responsive promoters from plant cells. In certain embodiments, the inducible promoter is a galactose-inducible promoter. In some embodiments, the inducible promoter is induced by one or more physiological conditions (e.g., pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, or concentration of one or more extrinsic or intrinsic inducing agents). Non-limiting examples of an extrinsic inducer or inducing agent include amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones or any combination thereof.

[0175] In some embodiments, the promoter is a constitutive promoter. As used in this application, a “constitutive promoter” refers to an unregulated promoter that allows continuous transcription of a gene. Non-limiting examples of a constitutive promoter include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, HXT3, HXT7, ACT1, ADH1, ADH2, ENO2, and SOD1.

[0176] Other inducible promoters or constitutive promoters known to one of ordinary skill in the art are also contemplated.

[0177] In some embodiments, introduction of a polynucleotide, such as a polynucleotide encoding an enzyme associated with the disclosure, into a host cell results in genomic integration of the polynucleotide. In some embodiments, a host cell (e.g., a yeast cell or a bacterial cell) comprises at least 1 copy, at least 2 copies, at least 3 copies, at least 4 copies, at least 5 copies, at least 6 copies, at least 7 copies, at least 8 copies, at least 9 copies, at least 10 copies, at least 11 copies, at least 12 copies, at least 13 copies, at least 14 copies, at least 15 copies, at least 16 copies, at least 17 copies, at least 18 copies, at least 19 copies, at least 20 copies, at least 21 copies, at least 22 copies, at least 23 copies, at least 24 copies, at least 25 copies, at least 26 copies, at least 27 copies, at least 28 copies, at least 29 copies, at least 30 copies, at least 31 copies, at least 32 copies, at least 33 copies, at least 34 copies, at least 35 copies, at least 36 copies, at least 37 copies, at least 38 copies, at least 39 copies, at least 40 copies, at least 41 copies, at least 42 copies, at least 43 copies, at least 44 copies, at least 45 copies, at least 46 copies, at least 47 copies, at least 48 copies, at least 49 copies, at least 50 copies, at least 60 copies, at least 70 copies, at least 80 copies, at least 90 copies, at least 100 copies, or more, including any values in between, of a polynucleotide sequence, such as a polynucleotide sequence encoding any of the polypeptides described in this application, in its genome. Said copies may be inserted into the same locus or into different loci of a recombinant host cell of the disclosure. 4)

[0178] In some embodiments, the sequence of a polynucleotide (e.g., a polynucleotide comprising a gene) is codon optimized. Codon optimization may increase expression of a gene by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, including all values in between relative to a reference sequence that is not codon-optimized.

[0179] In some embodiments, a polynucleotide encoding an enzyme associated with the present disclosure comprises a sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%, including all values in between, identical to any one of SEQ ID NOs: 14-29. In certain embodiments, a polynucleotide encoding an enzyme associated with the present disclosure comprises the sequence of any one of SEQ ID NOs: 14-29. In certain embodiments a polynucleotide encoding an enzyme associated with the present disclosure consists of or consists essentially of the sequence of any one of SEQ ID NOs: 14-29.

[0180] The polynucleotides disclosed herein can comprise a nucleotide sequence of any one of SEQ ID NOs: 14-29. In some instances, the polynucleotide comprises a variant of a nucleotide sequence of any one of SEQ ID NOs: 14-29. For instance, the polynucleotide can comprise a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% sequence identity to a nucleotide sequence set forth as any one of SEQ ID NOs: 14-29.

[0181] It would be understood that the host cells disclosed herein, e.g., S. boulardii encoding an AroZ or variant thereof, can comprise one or more than one of any of the heterologous polynucleotides disclosed above. In some instances, the host cell comprises two polynucleotides encoding the same protein (e.g., Arol, Aro3, or Aro4), wherein said polynucleotides sequences differ by at least one nucleotide. In such instances, the polynucleotide sequences can share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. It would be understood that polynucleotide sequence having less than 100% sequence identity can encode a protein (e.g., an enzyme disclosed herein) having the same amino acid sequence. In some instances, polynucleotide sequences having less than 100% sequence identity encode proteins having less than 100% sequence identity. Variant enzymes that can be encoded by the polynucleotides are disclosed herein. 4)

[0182] Host Cells

[0183] Any of the polynucleotides or polypeptides of the disclosure may be expressed in a host cell. As used in this application, the term “host cell” refers to a cell that is capable of expressing a polynucleotide, such as a polynucleotide that encodes a polypeptide used in production of PCA, precursors thereof, and products for which PCA is a precursor. The polynucleotide can be a heterologous polynucleotide.

[0184] Any suitable host cell may be used to express any of the recombinant polypeptides and other polypeptides disclosed in this application, including eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, fungal cells (e.g., yeast cells), bacterial cells (e.g., E. coli cells), algal cells, plant cells, insect cells, and animal cells, including mammalian cells.

[0185] Suitable yeast host cells include, but are not limited to: Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the yeast cell is Hansenula polymorpha, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces cerevisiae CEN. PK, Saccharomyces cerevisiae var. boulardii, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

[0186] In some embodiments, the yeast cell is Saccharomyces cerevisiae var. boulardii. In the present disclosure, the yeast cell name Saccharomyces cerevisiae var. boulardii is used interchangeably with the yeast cell name Saccharomyces boulardii (i.e., S. boulardii).

[0187] In some embodiments, the yeast strain is an industrial polyploid yeast strain. Other nonlimiting examples of fungal cells include cells obtained from Aspergillus spp., Penicillium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.

[0188] In some embodiments, the host cell is an algal cell such as Chlamydomonas (e.g., C.

[0189] Reinhardtii) and Phormidium (P. sp. ATCC29409).

[0190] In other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include gram positive, gram negative, and gram-variable bacterial cells. The host cell may be a species of, but not limited to: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azotobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, 4)

[0191] Campestris, Campylobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Saccharopolyspora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas.

[0192] In some embodiments, the bacterial host strain is an industrial strain. Numerous bacterial industrial strains are known and suitable for the methods and compositions described in this application.

[0193] In some embodiments, the bacterial host cell is of the Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, A. rubi), the Arthrobacter species (e.g, A. aurescens, A. citreus, A. globformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophormiae, A. roseoparaffinus, A. sulfureus, A. ureafaciens), the Bacillus species (e.g., B. thuringiensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulars, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, B. amyloliquefaciens). In particular embodiments, the host cell will be an industrial Bacillus strain including but not limited to B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausii, B. stearothermophilus and B. amyloliquefaciens. In some embodiments, the host cell will be an industrial Clostridium species (e.g, C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, C. beijerinckii). In some embodiments, the host cell will be an industrial Corynebacterium species (e.g., C. glutamicum, C. acetoacidophilum). In some embodiments, the host cell will be an industrial Escherichia species (e.g., E. coli). In some embodiments, the host cell will be an industrial Erwinia species (e.g, E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, E. terreus). In some embodiments, the host cell will be an industrial Pantoea species (e.g, P. citrea, P. agglomerans). In some embodiments, the host cell will be an industrial Pseudomonas species, (e.g., P. putida, P. aeruginosa, P. mevalonii). In some embodiments, the host cell will be an

[0194] industrial Streptococcus species (e.g, S. equisimiles, S. pyogenes, S. uberis). In some embodiments, the host cell will be an industrial Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. 4)

[0195] avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, S. Hvidans), In some embodiments, the host cell will be an industrial Zymomonas species (e.g., Z. mobilis, Z. lipolytica).

[0196] The present disclosure is also suitable for use with a variety of animal cell types, including mammalian cells, for example, human (including 293, HeLa, WI38, PER. C6 and Bowes melanoma cells), mouse (including 3T3, NSO, NS1, Sp2 / 0), hamster (CHO, BHK), monkey (COS, FRhL, Vero), and hybridoma cell lines.

[0197] In various embodiments, cell types or strains that may be used in the practice of the disclosure including both prokaryotic and eukaryotic cell or strains, and are readily accessible to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0198] Any host cell encoding enzymes necessary to produce PCA when a heterologous polynucleotide encoding a 3-dehydroshikimate dehydratase (AroZ) or variant thereof is introduced into said cell is envisaged as being within the scope of the host cells described herein. In particular, any host cell which is capable of producing 3-dehydroshikimate (3-DHS) and lacks an enzyme capable of converting 3-DHS to PCA (e.g., 3-dehydroshikimate dehydratase) is envisaged within the scope of the host cells disclosed herein. In such instances, the host cells can comprise any cell capable of converting pyruvate to 3-DHS, and lacks an enzyme capable of converting 3-DHS to PCA (e.g., 3-dehydroshikimate dehydratase). In another instance, a host cell comprises a functional shikimic acid pathway and lacks an enzyme capable of converting 3-DHS to PCA (e.g., 3-dehydroshikimate dehydratase). In some embodiments, a host cell can be any host cell which is capable of producing PCA following introduction of a heterologous polynucleotide encoding a 3-dehydroshikimate dehydratase (AroZ) or variant thereof into the cell.

[0199] The term “cell,” as used in this application, may refer to a single cell or a population of cells, such as a population of cells belonging to the same cell line or strain. Use of the singular term “cell” should not be construed to refer explicitly to a single cell rather than a population of cells. The host cell may comprise genetic modifications relative to a wild-type counterpart.

[0200] A vector or polynucleotide encoding any one or more of the polypeptides described in this application may be introduced into a suitable host cell using any method known in the art. Host cells may be cultured under any conditions suitable as would be understood by one of ordinary skill in the art. For example, any media, temperature, and incubation conditions known in the art may be used. 4)

[0201] For host cells carrying an inducible vector, cells may be cultured with an appropriate inducible agent to promote expression.

[0202] Any of the cells disclosed in this application can be cultured in media of any type (rich or minimal) and any composition prior to, during, and / or after contact and / or integration of a nucleic acid. The conditions of the culture or culturing process can be optimized through routine experimentation as would be understood by one of ordinary skill in the art. In some embodiments, the selected media is supplemented with various components. In some embodiments, the concentration and amount of a supplemental component is optimized. In some embodiments, other aspects of the media and growth conditions (e.g., pH, temperature, etc.) are optimized through routine experimentation. In some embodiments, the frequency that the media is supplemented with one or more supplemental components, and the amount of time that the cell is cultured, is optimized.

[0203] Culturing of the cells described in this application can be performed in culture vessels known and used in the art. In some embodiments, an aerated reaction vessel (e.g., a stirred tank reactor) is used to culture the cells. In some embodiments, a bioreactor or fermenter is used to culture the cells. Thus, in some embodiments, the cells are used in fermentation. As used in this application, the terms “bioreactor” and “fermenter” are interchangeably used and refer to an enclosure, or partial enclosure, in which a biological, biochemical and / or chemical reaction takes place, involving a living organism or part of a living organism, or purified proteins. Any type of bioreactor or fermenter known in the art may be compatible with aspects of the disclosure.

[0204] In some embodiments, a host cell that expresses a heterologous polynucleotide encoding an enzyme or variant thereof disclosed herein may increase production of PC A, one or more precursors of PC A, or a product for which PC A is a precursor by approximately 1.1 -fold, 1.2-fold, 1.3 -fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, or 6-fold more (e.g., 2-fold to 6-fold more) relative to a control. In some embodiments, the control is a host cell that expresses a wildtype AroZ, Arol, Aro3, Aro4, Tkll, Tall, AroB, AroD, and / or AroF.

[0205] In some embodiments, a host cell expresses a heterologous polynucleotide encoding a variant enzyme that comprises an amino acid sequence that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 4)

[0206] least 98%, at least 99%, or is 100% identical to any one of SEQ ID NOs: 1-13 or an enzyme otherwise described in this disclosure. In some embodiments, the amino acid sequence of an enzyme associated with the present disclosure comprises or consists of any one of SEQ ID NOs: 1-13, or a conservatively substituted version thereof.

[0207] Disclosed herein are host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1. In some instances, the AroZ variant comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. In some instances, the AroZ variant comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. In some instances, the AroZ variant comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. In some instances, the AroZ variant comprises an amino acid sequence set forth as SEQ ID NO: 1. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0208] Host cells are disclosed herein. The host cell can be any host cell disclosed herein. For instance, the host cell can be a yeast cell, such as Saccharomyces. In such instances, the yeast cell can be a Saccharomyces cerevisiae cell or a Saccharomyces cerevisiae var. boulardii cell.

[0209] The host cells can further comprise at least a second heterologous polynucleotide sequence encoding each of the following genes: arol, aro3, aro4, tkll, and tall. For instance, the host cell can comprise a second heterologous polynucleotide one of the following genes: arol, aro3, aro4, tkll, and tall. In such instances, the host cell can comprise a third heterologous polynucleotide one of the following genes: arol, aro3, aro4, tkll, and tall. In such instances, the host cell can comprise a fourth heterologous polynucleotide one of the following genes: arol, aro3, aro4, tkll, and tall. In such instances, the host cell can comprise a fifth heterologous polynucleotide one of the following genes: arol, aro3, aro4, tkll, and tall. In such instances, the host cell can comprise a sixth heterologous polynucleotide one of the following genes: arol, aro3, aro4, tkll, and tall. In such instances, the host cell can comprise a seventh heterologous polynucleotide one of the following genes: arol, aro3, aro4, tkll, and tall. In such instances, the host cell can comprise an eighth heterologous polynucleotide one of the following genes: arol, aro3, aro4, tkll, and tall. 4)

[0210] Host cells can comprise at least two copies of any genes disclosed herein. For instance, host cells can comprise at least two copies of any gene that contributes to PCA production in the host cell. For instance, the host cell can comprise at least two copies of any one of the following genes: arol, aro3, aro4, tkll, and tall. In such instances, the host cell can comprise at least two copies of an arol gene. In some instances, the host cell can comprise at least two copies of an aro3 gene. In some instances, the host cell can comprise at least two copies of an aro4 gene. In some instances, the host cell can comprise at least two copies of a tkll gene. In some instances, the host cell can comprise at least two copies of a tall gene. A host cell may comprise more than two copies of any one of the genes disclosed herein. It would be understood that host cells can have two or more copies of multiple genes. For instance, a host cell may have two or more copies of at least two gene selected from the following genes: arol, aro3, aro4, tkll, and tall.

[0211] Host cells disclosed herein may comprise at least two copies of the following genes: arol and aro3. In some instances, the host cell comprises two copies of an arol gene. In some instances, the host cell comprises two copies of an aro3 gene. In such instances, the host cell can comprise two copies of an arol and an aro3 gene.

[0212] Any of the heterologous polynucleotides present within a host cell may be codon-optimized. It would be understood that any individual polynucleotide may be codon-optimized, while not all heterologous polynucleotides are codon-optimized within a host cell.

[0213] Disclosed herein are Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The some instances, the variant comprises 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The Saccharomyces cerevisiae host cells can comprise a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0214] In some instances, the Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid 4)

[0215] sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, further comprises a heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3. In such instances, the Arol variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3. In some instances, the Saccharomyces cerevisiae host cell comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, and a heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3. The Arol enzyme can be encoded by a polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16 or 27, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 16 or 27. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0216] In some instances, the Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, further comprises a heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11. In such instances, the Arol variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11. In some instances, the Saccharomyces cerevisiae host cell comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, and a heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11. The Arol enzyme can be encoded by a polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 24, or a variant thereof having at least 80%, at least 85%, 4)

[0217] at least 90%, at least 95%, or at least 99% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 24. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0218] In some instances, the Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, further comprises a heterologous polynucleotide encoding a first copy of an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11, and a second copy of an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3. In such instances, the first copy of the Arol variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11. In such instances, the second copy of the Arol variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0219] In some instances, the Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, further comprises a heterologous polynucleotide encoding an Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4. In such instances, the Aro3 variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4. In some instances, the Saccharomyces 4)

[0220] cerevisiae host cell comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, and a heterologous polynucleotide encoding an Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4. The Aro3 enzyme can be encoded by a polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 17 or 28, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 17 or 28. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0221] In some instances, the Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, further comprises a heterologous polynucleotide encoding an Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12. In such instances, the Aro3 variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12. In some instances, the Saccharomyces cerevisiae host cell comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, and a heterologous polynucleotide encoding an Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12. The Aro3 enzyme can be encoded by a polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 25, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 25. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0222] In some instances, the Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, further comprises a heterologous 4)

[0223] polynucleotide encoding a first copy of an Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and a second copy of an Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12. In such instances, the first copy of the Aro3 variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4. In such instances, the second copy of the Aro3 variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0224] In some instances, the Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, further comprises a heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 5. In such instances, the Aro4 variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 5. In some instances, the Saccharomyces cerevisiae host cell comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, and a heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5. The Aro4 enzyme can be encoded by a polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 18 or 29, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the nucleotide sequence set forth as SEQ 4)

[0225] ID NO: 18 or 29. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0226] In some instances, the Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, further comprises a heterologous polynucleotide encoding an Tkl1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6. In such instances, the Tkl1 variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6. In some instances, the Saccharomyces cerevisiae host cell comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, and a heterologous polynucleotide encoding an Tkl1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6. The Tkl1 enzyme can be encoded by a polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 19, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 19. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0227] In some instances, the Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, further comprises a heterologous polynucleotide encoding an Tal1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7. In such instances, the Tal1 variant can comprise 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7. In some instances, the Saccharomyces 4)

[0228] cerevisiae host cell comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, and a heterologous polynucleotide encoding an Tal1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7. The Tal1 enzyme can be encoded by a polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 20, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 20. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0229] In some instances, the Saccharomyces cerevisiae host cells comprise a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein said host cell further comprises the following heterologous polynucleotides encoding codon- optimized variants: i) a second heterologous polynucleotide encoding an Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkl1 enzyme comprising an amino acid sequence set 4)

[0230] forth as SEQ ID NO: 6; and / or vii) an eighth heterologous polynucleotide encoding a Tal1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0231] Disclosed herein are Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, wherein said host cell further comprises the following heterologous polynucleotides encoding codon-optimized variants: i) a second heterologous polynucleotide encoding an Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkl1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; or vii) an eighth heterologous polynucleotide encoding a Tal1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0232] In some instances, the Saccharomyces cerevisiae host cells comprise a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein said host cell further comprises the following heterologous polynucleotides encoding codon- optimized variants: i) a second heterologous polynucleotide encoding an Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11 or a variant thereof comprising an amino acid 4)

[0233] sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkl1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; or vii) an eighth heterologous polynucleotide encoding a Tal1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0234] In some instances, the Saccharomyces cerevisiae host cells comprise a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein said host cell further comprises the following heterologous polynucleotides encoding codon- optimized variants: i) a second heterologous polynucleotide encoding an Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous 4)

[0235] polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkl1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; and vii) an eighth heterologous polynucleotide encoding a Tal1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7 or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0236] Disclosed herein are Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, wherein said host cell further comprises the following heterologous polynucleotides encoding codon-optimized variants: i) a second heterologous polynucleotide encoding an Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkl1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; and / or vii) an eighth heterologous polynucleotide encoding a Tal1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. 4)

[0237] Disclosed herein are Saccharomyces cerevisiae host cells comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, wherein said host cell further comprises the following heterologous polynucleotides encoding codon-optimized variants: i) a second heterologous polynucleotide encoding an Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Aro1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkl1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; and vii) an eighth heterologous polynucleotide encoding a Tal1 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7. In certain embodiments, a strain with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0238] Cultivation of S. boulardii

[0239] Populations or cultures of S. boulardii can be produced by cultivation of a specific yeast strain. Methods of cultivating yeast are readily known in the art. For instance, materials and methods for the successful large-scale cultivation of Saccharomyces cerevisiae var. boulardii probiotic yeast production are described in Moonsamy, G. et al., Development of a High-Cell-Density Production Process for a Biotherapeutic Yeast, Saccharomyces cerevisiae var. boulardii, for Use as a Human Probiotic. Fermentation 2025, 11, 186, the contents of which are incorporated herein by reference in their entirety. Cultivation can be started by scaling-up a seed culture. This involves repeatedly and aseptically transferring the culture to a larger and larger volume to serve as the inoculum for the fermentation, which can be carried out in large stainless steel fermenters in medium containing proteins, carbohydrates, and minerals necessary for optimal growth of the strain. Media for cultivating yeast are readily known in the art and commercially available. Any medium known in the art can be used to cultivate S. boulardii. Non-limiting exemplary media include yeast extract peptone dextrose (YPD) broth and Sabouraud Dextrose Agar (SDA). In some instances, specialized media can be used, for instance, to emulate specific growth environments. Such mediums are commercially available and readily known in the art. After the yeast inoculum is added to the fermentation vessel, the temperature 4)

[0240] and agitation are controlled to allow maximum growth. Once the culture reaches a maximum population density, the culture is harvested by separating the cells from the fermentation medium (i.e., supernatant). This separation is commonly performed by centrifugation. Subsequent filtration can be used to remove intact yeast cells. In such instances, a cell-free culture supernatant (CFCS) can be isolated.

[0241] The concentration of the yeast culture can be measured from any sample of fermentation broth or yeast strain composition. A colony forming unit (CFU) is the viable cell count of a sample resulting from standard microbiological plating methods. The term is derived from the fact that a single cell when plated on appropriate medium will grow and become a viable colony in the agar medium. Since multiple cells may give rise to one visible colony, the term colony forming unit can be a more useful unit measurement than cell number.

[0242] The microbial compositions disclosed herein can comprise an amount of Saccharomyces cerevisiae var. boulardii comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or comprises a conservatively substituted version of SEQ ID NO: 1. Such amount can comprise a concentration of Saccharomyces cerevisiae var. boulardii of at least about 104CFU / g to about 1012CFU / g, about 105CFU / g to about 1012CFU / g, about 106CFU / g to about 1012CFU / g, about 107CFU / g to about 1012CFU / g, about 108CFU / g to about 1012CFU / g, about 109CFU / g to about 1012CFU / g, about IO10CFU / g to about 1012CFU / g, about 1011CFU / g to about 1012CFU / g, about 105CFU / g to about 1011CFU / g, about 105CFU / g to about 1011CFU / g, about 106CFU / g to about 1011CFU / g, about 107CFU / g to about 1011CFU / g, about 108CFU / g to about 1011CFU / g, about 109CFU / g to about 1011CFU / g, about IO10CFU / g to about 1011CFU / g, about 105CFU / g to about IO10CFU / g, about 106CFU / g to about IO10CFU / g, about 107CFU / g to about IO10CFU / g, about 108CFU / g to about IO10CFU / g, or about 109CFU / g to about IO10CFU / g. In other embodiments, the concentration of Saccharomyces cerevisiae var. boulardii comprises or consists of at least about 104CFU / g, at least about 105CFU / g, at least about 106CFU / g, at least about 107CFU / g, at least about 108CFU / g, at least about 109CFU / g, at least about IO10CFU / g, at least about 1011CFU / g, or at least about 1012CFU / g. Another such amount can comprise a concentration of Saccharomyces cerevisiae var. boulardii of at least about 104CFU / ml to about 1012CFU / ml, about 105CFU / ml to about 1012CFU / ml, about 106CFU / ml to about 1012CFU / ml, about 107CFU / ml to about

[0243] 1012CFU / ml, about 108CFU / ml to about 1012CFU / ml, about 109CFU / ml to about 1012CFU / ml, 4)

[0244] about IO10CFU / ml to about 1012CFU / ml, about 1011CFU / ml to about 1012CFU / ml, about

[0245] 105CFU / ml to about 1011CFU / ml, about 105CFU / ml to about 1011CFU / ml, about 106CFU / ml to about 1011CFU / ml, about 107CFU / ml to about 1011CFU / ml, about 108CFU / ml to about

[0246] 1011CFU / ml, about 109CFU / ml to about 1011CFU / ml, about IO10CFU / ml to about 1011CFU / ml, about 105CFU / ml to about IO10CFU / ml, about 106CFU / ml to about IO10CFU / ml, about 107CFU / ml to about IO10CFU / ml, about 108CFU / ml to about IO10CFU / ml, or about 109CFU / ml to about IO10CFU / ml. In other embodiments, the concentration of Saccharomyces cerevisiae var. boulardii comprises or consists of at least about 104CFU / ml, at least about 105CFU / ml, at least about 106CFU / ml, at least about 107CFU / ml, at least about 108CFU / ml, at least about 109CFU / ml, at least about IO10CFU / ml, at least about 1011CFU / ml, or at least about 1012CFU / ml.

[0247] It would be understood that the microbial compositions described above could comprise a probiotic composition.

[0248] Disclosed herein are microbial compositions comprising at least a first host cell and / or cell constituent(s) thereof, wherein said at least first host cell comprises a Saccharomyces cerevisiae var. boulardii (S. boulardii) capable of producing PCA. Microbial compositions disclosed herein can comprise at least a first host cell, and / or cell constituent(s) thereof, wherein said at least first host cell comprises S. boulardii comprising a heterologous polynucleotide encoding a 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or comprises a conservatively substituted version of SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. Such microbial compositions can further comprise PCA. PCA can be extracellular to said S. boulardii (e.g., wherein said PCA has been secreted into a supernatant). PCA can be present at any concentration. In some instances, the microbial composition comprises an effective amount of PCA. An “effective amount” when used in the context of PCA refers to any quantity of PCA which achieves a desired result of any method described herein. In particular, an effective amount of PCA can maintain and / or alter at least one biological activity in a subject following administration of a microbial composition.

[0249] In some instances, PCA is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the microbial composition weight per volume (w / v), or volume per volume ( v / v). In some instances, PCA is present at a concentration of at least 0.0005 mM (i.e., 0.5 gM), 0.001 mM (i.e., 1 uM), at least 0.005 4)

[0250] mM, at least 0.01 M, at least 0.03 M, at least 0.06 mM, at least 0.12 mM, at least 0.25 mM, at least 0.5 mM, at least 1 mM, at least 2 mM, at least 5 M, or more than 5 mM. In certain instances, PCA is present at a concentration of between about 0.5 pM to about 5 pM (i.e, 0.005 mM), about 1 uM to about 10 uM (i.e., 0.01 mM), about 0.005 mM to about 0.03 mM, about 0.01 mM to about 0.06 mM, about 0.03 mM to about 0.12 mM, or about 0.06 mM to about 0.25 mM.

[0251] PCA can be present in the Saccharomyces cerevisiae var. boulardii microbial compositions at a concentration of between about 0.001 mM to about 0.01 mM, about 0.002 mM to about 0.01 mM, about 0.003 mM to about 0.01 mM, about 0.004 mM to about 0.01 mM, about 0.005 mM to about 0.01 mM, about 0.006 mM to about 0.01 mM, about 0.007 mM to about 0.01 mM, about 0.008 mM to about 0.01 mM, or about 0.009 mM to about 0.01 mM. In other instances, PCA is present at a concentration of between about 0.01 mM to about 0.03 mM, about 0.011 mM to about 0.03 mM, about 0.012 mM to about 0.03 mM, about 0.013 mM to about 0.03 mM, about 0.014 mM to about 0.03 mM, about 0.015 mM to about 0.03 mM, about 0.016 mM to about 0.03 mM, about 0.017 mM to about 0.03 mM, about 0.018 mM to about 0.03 mM, about 0.019 mM to about 0.03 mM, about 0.02 mM to about 0.03 mM, about 0.022 mM to about 0.03 mM, about 0.023 mM to about 0.03 mM, about 0.024 mM to about 0.03 mM, about 0.025 mM to about 0.03 mM, about 0.026 mM to about 0.03 mM, about 0.027 mM to about 0.03 mM, about 0.028 mM to about 0.03 mM, about 0.029 mM to about 0.03 mM.

[0252] In some instances, PCA is present at a concentration of about 0.12 mM. In some instances, PCA is present at a concentration of about 0.03 mM. In some instances, PCA is present at a concentration of about 0.01 mM. In some instances, PCA is present at a concentration of about 0.005 mM.

[0253] The microbial compositions comprising S. boulardii and / or cell constituents ) thereof may comprise PCA at any concentration disclosed herein. In some instances, the microbial composition is a S’. boulardii cell culture. In some instances, the microbial composition is a S. boulardii fermentation culture. In some instances, the microbial composition comprises a formulated microbial composition. In such instances, the microbial composition comprises a food and / or beverage product. Such microbial formulations can comprise between 2 g / L to 4 g / L, between 3 g / L to 5 g / L, between 4 g / L to 6 g / L, between 5 g / L to 7 g / L. between 6 g / L to 8 g / L, between 7 g / L to 9 g / L, between 8 g / L to 10 g / L, between 9 g / L to 11 g / L, between 10 g / L to 12 g / L, between 11 g / L to 13 g / L, between 12 g / L to 14 g / L, between 13 g / L to 15 g / L, between 14 g / L to 16 g / L, between 15 g / L to 17 g / L, between 16 g / L to 18 g / L, between 17 g / L to 19 g / L, between 18 g / L to 20 g / L, between 19 g / L to 21 g / L, between 20 g / L to 22 g / L, between 21 g / L to 23 g / L, between 22 g / L to 24 g / L, between 23 g / L to 25 g / L, between 4)

[0254] 24 g / L to 26 g / L, between 25 g / L to 27 g / L, between 26 g / L to 28 g / L, between 27 g / L to 29 g / L, between 28 g / L to 30 g / L, between 29 g / L to 31 g / L, between 30 g / L to 32 g / L, between 31 g / L to 33 g / L, between 32 g / L to 34 g / L, between 33 g / L to 35 g / L, between 34 g / L to 36 g / L, between 35 g / L to 37 g / L, between 36 g / L to 38 g / L, between 37 g / L to 39 g / L, between 38 g / L to 40 g / L, between 39 g / L to 41 g / L, between 40 g / L to 42 g / L, between 41 g / L to 43 g / L, between 42 g / L to 44 g / L, between 43 g / L to 45 g / L, between 44 g / L to 46 g / L, between 45 g / L to 47 g / L, between 46 g / L to 48 g / L, between 47 g / L to 49 g / L, between 48 g / L to 50 g / L, between 49 g / L to 51 g / L, between 50 g / L to 52 g / L, between 51 g / L to 53 g / L, between 52 g / L to 54 g / L, between 53 g / L to 55 g / L, between 54 g / L to 56 g / L, between 55 g / L to 57 g / L, between 56 g / L to 58 g / L, between 57 g / L to 59 g / L, between 58 g / L to 60 g / L, or more than 60 g / L of PCA. In some embodiments, the composition comprises a liquid composition (e.g., a cell culture or beverage product). In some instances, the microbial composition comprising S. boulardii and / or cell constituent(s) thereof comprises PCA at a concentration of at least 2 g / L, at least 4 g / L, at least 6 g / L, at least 8 g / L, at least 10 g / L, at least 12 g / L, at least 14 g / L, at least 16 g / L, at least 18 g / L, at least 20 g / L, at least 22 g / L, at least 24 g / L, at least 26 g / L, at least 28 g / L, at least 30 g / L, at least 32 g / L, at least 34 g / L, at least 36 g / L, at least 38 g / L, at least 40 g / L, at least 42 g / L, at least 44 g / L, at least 46 g / L, at least 48 g / L, at least 50 g / L, at least 52 g / L, at least 54 g / L, at least 56 g / L, at least 58 g / L, at least 60 g / L.

[0255] In some instances, PCA is present in the microbial composition at any concentration depicted in Tables 9-11.

[0256] The microbial compositions disclosed herein can be administered to any tissue of a subject. As such, the microbial compositions can further comprise cells and / or tissue of said subject. In particular, the microbial compositions disclosed herein can further comprise i) luminal contents of the gastrointestinal tract of a subject; ii) at least one intestinal epithelial cell of a subject, or iii) or at least one skin epithelial cell of a subject.

[0257] Disclosed herein are microbial compositions comprising a plurality of Saccharomyces cerevisiae var. boulardii (S. boulardii) and / or cell constituent(s) thereof, wherein said S. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of SEQ ID NO: 1. In some instances, the variant comprises 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 4)

[0258] least 98%, at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The Saccharomyces cerevisiae host cells can comprise a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase ( AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1.

[0259] The S. boulardii present in the microbial compositions can comprise any additional heterologous polynucleotide disclosed herein. For instance, the S. boulardii can comprise one or more heterologous polynucleotides, wherein said one or more heterologous polynucleotides comprise a heterologous polynucleotide sequence encoding any of the following genes: arol, aro3, aro4, tkll, and tall.

[0260] In some instances, the microbial composition comprises a plurality of S. boulardii and / or cell constituent(s) thereof, wherein said S. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, wherein said S. boulardii further comprises the following heterologous polynucleotides encoding codon-optimized variants: i) a second heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkll enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; and vii) an eighth heterologous polynucleotide encoding a Tall enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7.

[0261] Fermentation

[0262] Postbiotic compositions disclosed herein can be generated following fermentation of S. boulardii. Fermentation of S. boulardii is an established method known in the art. In particular, conditions and medium suitable for S. boulardii fermentation are readily understood to one of ordinary skill in the art. Any suitable conditions (e.g., medium and / or environmental conditions) are envisaged as suitable for fermentation of the S. boulardii described herein. Fermentation may include regulating various factors, such as pH levels, temperature, moisture, oxygen concentration (or lack thereof), and / or duration of time. In some embodiments, fermentation may include the introduction of a culture 4)

[0263] media or growth media. Such additional culture media (i.e., feed medium) may aid in the growth and metabolic activity of S. boulardii, and / or production of a desired postbiotic agent. In some embodiments, fermentation may include the introduction of one or more culture media.

[0264] Fermentation can involve agitation of a fermentation culture. In some embodiments, the step of fermenting occurs for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 or more than 23 hours. In some embodiments, the culture is agitated at a temperature of 37°C. In some embodiments, the step of fermenting involves agitating at a temperature of 37°C.

[0265] The step of fermenting can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24 or more than 24 hours. For instance, the step of fermenting is about 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, 7-8 hours, 8-9 hours, 9-10 hours, 10-15 hours, 15-20 hours, 20-24 hours, or more than 24 hours. In some embodiments, the step of fermenting is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 hours. In some embodiments, the step of fermenting is 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, 7-8 hours, 8-9 hours, 9-10 hours, 10-15 hours, 15-20 hours, 20-24 hours, or more than 24 hours. Fermentation of the disclosed S. boulardii can be longer than 24 hours. For instance, S. boulardii can be incubated under suitable fermentative growth conditions for at least 24, at least 36, at least 48, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, or more than 120 hours. In such instances, S. boulardii can be incubated under suitable fermentative growth conditions for between 12-36 hours, between 24-48 hours, between 36-60 hours, between 48-72 hours, between 60-84 hours, between 72-96 hours, between 84-108 hours, or between 96-120 hours. For instance, S. boulardii can be incubated under suitable fermentative growth conditions for about 24, about 36, about 48, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, about 120 hours, or more than 120 hours. Any improvement over these would be unexpected. Anything elese

[0266] Fermentation is performed at any temperature suitable for S. boulardii. In one aspect, fermentation is performed at between about 28°C to about 32°C. In another aspect, fermentation is performed at between about 29°C to about 31°C. In another aspect, fermentation is performed at between about 29.5°C to about 30.5°C. In another aspect, fermentation is performed at about 30°C. 4)

[0267] Fermentation can be performed in a fermentation vessel at any volume. In some instances, the volume of the fermentation culture can be at least 0.5 L (liters), at least 1 L, at least 2 L, at least 3 L, at least 4 L, at least 5 L, at least 6 L, at least 7 L, at least 8 L, at least 9 L, at least 10 L, at least 11 L, at least 12 L, at least 13 L, at least 14 L, at least 15 L, at least 16 L, at least 17 L, at least 18 L, at least 19 L, at least 20 L, or more than 20 L. In some instances, the volume of the fermentation culture is between about 10 L to about 10,000 liters (L). For example, the fermentation culture can be between 1-10 L, 5-50 L, 10-100 L, 50-500 L, 100-1000 L, 500-5,000 L, 1,000 L-10,000 L. In some instances, the volume of milk product can be greater than 10,000 L.

[0268] Disclosed herein are liquid cultures of S. boulardii under conditions suitable for fermentative growth. In some instances, a plurality of S. boulardii is undergoing fermentation growth. One of ordinary skill in the art would readily understand how to determine if a plurality of S. boulardii is undergoing fermentation growth. For instance, measurements of the culture may be taken and assessed (e.g., culture pH, glucose consumption, ethanol concentration). Liquid cultures (e.g., fermentation cultures) of S. boulardii may comprise PCA. Such cultures can be any volume described herein. The cultures comprising S. boulardii and / or cell constituent(s) thereof may comprise PCA at any concentration disclosed herein. In some instances, the cultures comprising S. boulardii and / or cell constituent(s) thereof comprises between 2 g / L to 4 g / L, between 3 g / L to 5 g / L, between 4 g / L to 6 g / L, between 5 g / L to 7 g / L, between 6 g / L to 8 g / L, between 7 g / L to 9 g / L, between 8 g / L to 10 g / L, between 9 g / L to 11 g / L, between 10 g / L to 12 g / L, between 11 g / L to 13 g / L, between 12 g / L to 14 g / L, between 13 g / L to 15 g / L, between 14 g / L to 16 g / L, between 15 g / L to 17 g / L, between 16 g / L to 18 g / L, between 17 g / L to 19 g / L, between 18 g / L to 20 g / L, between 19 g / L to 21 g / L, between 20 g / L to 22 g / L, between 21 g / L to 23 g / L, between 22 g / L to 24 g / L, between 23 g / L to 25 g / L, between 24 g / L to 26 g / L, between 25 g / L to 27 g / L, between 26 g / L to 28 g / L, between 27 g / L to 29 g / L, between 28 g / L to 30 g / L, between 29 g / L to 31 g / L, between 30 g / L to 32 g / L, between 31 g / L to 33 g / L, between 32 g / L to 34 g / L, between 33 g / L to 35 g / L, between 34 g / L to 36 g / L, between 35 g / L to 37 g / L, between 36 g / L to 38 g / L, between 37 g / L to 39 g / L, between 38 g / L to 40 g / L, between 39 g / L to 41 g / L, between 40 g / L to 42 g / L, between 41 g / L to 43 g / L, between 42 g / L to 44 g / L, between 43 g / L to 45 g / L, between 44 g / L to 46 g / L, between 45 g / L to 47 g / L, between 46 g / L to 48 g / L, between 47 g / L to 49 g / L, between 48 g / L to 50 g / L, between 49 g / L to 51 g / L, between 50 g / L to 52 g / L, between 51 g / L to 53 g / L, between 52 g / L to 54 g / L, between 53 g / L to 55 g / L, between 54 g / L to 56 g / L, between 55 g / L to 57 g / L, between 56 g / L to 58 g / L, between 57 g / L to 59 g / L, between 58 g / L to 60 g / L, or more than 60 g / L of PCA. In some embodiments, the cultures comprising S. boulardii and / or 4)

[0269] cell constituent(s) thereof comprises PCA at a concentration of at least 2 g / L, at least 4 g / L, at least 6 g / L, at least 8 g / L, at least 10 g / L, at least 12 g / L, at least 14 g / L, at least 16 g / L, at least 18 g / L, at least 20 g / L, at least 22 g / L, at least 24 g / L, at least 26 g / L, at least 28 g / L, at least 30 g / L, at least 32 g / L, at least 34 g / L, at least 36 g / L, at least 38 g / L, at least 40 g / L, at least 42 g / L, at least 44 g / L, at least 46 g / L, at least 48 g / L, at least 50 g / L, at least 52 g / L, at least 54 g / L, at least 56 g / L, at least 58 g / L, at least 60 g / L.

[0270] In some instances, PCA is present in a S. boulardii fermentation culture composition at any concentration depicted in Tables 9-11.

[0271] During fermentation, the pH of the S. boulardii culture may decrease, resulting in a culture having a lower pH compared to the initial culture. In some instances, the pH of the culture remains about the same during fermentation. In some instances, the initial pH of the S. boulardii culture during fermentation is between pH 4-6. In some instances, the initial pH of the culture is between 4.5 -6.5. In some instances, the initial pH of the culture is between 4.5-5.5. In some instances, the S. boulardii culture has an initial pH at or below pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, or pH 4.9. In some instances, the S. boulardii culture has a final pH at or below pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, or pH 4.5. In some instances, the final pH is between 4.5-5.5. In some instances, the final pH is between 4.6-5.5. In some instances, the final pH is between 4.7-5.5. In some instances, the final pH is between 4.8-5.5. In some instances, the final pH is between 4.9-5.5. The pH of the medium can be measured during fermentation. The pH can be measured at any point in time throughout fermentation. Methods of measuring pH of a medium are readily known in the art. Any method known in the art for measuring pH of a fermentation medium can be used. In some embodiments, fermentation is maintained until a specific pH value of the medium is achieved.

[0272] Colony forming units (CFUs). CFU(s) can be measured during fermentation. For instance, CFUs can be used to determine the concentration of microorganisms within a fermentation medium. Fermentation may be maintained until a target concentration of 5. boulardii is achieved. In some embodiments, fermentation is maintained until a concentration of at least about 104CFU / ml, at least about 10’ CFU / ml, at least about IO6CFU / ml, at least about 10' CFU / ml, at least about 10sCFU / ml, at least about IO CFLT / ml, at least about 10s0CFU / ml, at least about 10nCFU / mL or at least about 1012CFU / ml is achieved. This may be performed directly after inoculation, some period of time after inoculation, at regular periods during fermentation, or at any point in time throughout fermentation. In such instances, at least about 104CFU / ml, at least about 105CFU / ml, at least about 106CFU / ml, at 4)

[0273] least about 107CFU / ml, at least about 108CFU / ml, at least about 109CFU / ml, at least about IO10CFU / ml, at least about 1011CFU / ml, or at least about 1012CFU / ml of S. boulardii can be viable cells present within a fermentation medium.

[0274] Various components can be added to a fermentation medium in order to enhance fermentation. For instance, culture media can be added in the form of a feed medium, wherein said culture media comprises carbon, nitrogen, minerals, amino acids, vitamins, or derivatives, or combinations thereof. Compositions comprising such components, and combinations thereof, and a plurality of S. boulardii and / or cell constituents thereof are envisaged within the scope of the disclosure. For instance, a fermentation culture can comprise a plurality of S. boulardii and / or cell constituents thereof, and a culture medium. Culture mediums useful for cultivation and fermentation of yeast, such as S. boulardii are well known in the art. A fermentation culture composition can comprise any known culture medium disclosed herein, e.g., yeast extract peptone dextrose (YPD), or components or derivatives thereof. Accordingly, a fermentation culture composition can comprise any components or derivatives thereof present within a culture medium suitable for cultivation and / or fermentative growth of S. boulardi. Such components can include carbon, nitrogen, minerals, amino acids, vitamins, or derivatives, or combinations thereof. A carbon source can include, but is not limited to, glucose, sucrose, fructose, glycerol, and / or starch hydrolysates. A nitrogen source can include, but is not limited to, ammonium salts, amino acids, yeast extract, or peptone. Salts can comprise, but are not limited to, one or more of phosphate, sulfate, magnesium, potassium, iron, and trace elements. A fermentation culture composition can further comprise a supplementation composition. A supplementation composition would be understood to enhance S. boulardii cultivation and / or fermentative growth. For instance, the addition of a supplementation composition may increase S. boulardii replication rate. In some instances, a supplementation composition may increase PCA production during S. boulardii cultivation and / or fermentative growth. A supplementation composition can comprise vitamins, buffering agents, surfactants, antifoaming agents, and / or inducers of metabolic pathways. Components and / or derivatives thereof are added to a fermentation culture at concentrations suitable for S. boulardii cultivation and / or fermentative growth. Such concentrations are well established in the art. Any concentration known to be suitable for S. boulardii cultivation and / or fermentation can be used.

[0275] In some embodiments, the step of fermenting may comprise “kicking” the fermentation process. “Kicking” may aid in consumption of culture media by the plurality of. boulardii in order to increase the concentration of S. boulardii in the medium. In some embodiments, kicking comprises the addition of fermentable sugars, wherein the fermentable sugars are consumed by a plurality of S. 4)

[0276] boulardii. In some embodiments, the fermentable sugars include any sugars as described in this disclosure. In some embodiments, the fermentable sugars comprise glucose or a derivative thereof. In some embodiments, kicking comprises agitating and / or mixing to increase the rate of fermentation.

[0277] Once produced, the S. boulardii culture can be cooled and stored at a temperature of around 4°C.

[0278] S. boulardii-derived postbiotic compositions

[0279] Disclosed herein are Saccharomyces cerevisiae var. boulardii (S. boulardii) -derived postbiotic compositions comprising a plurality of S. boulardii, and / or cell constituent(s) thereof. Any strain of S. boulardii described herein can be used to produce a postbiotic composition. For instance, the S. boulardii can comprise a heterologous polynucleotide encoding a 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or comprises a conservatively substituted version of SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. Postbiotic compositions described can be produced following cultivation of yeast strains. Production of a postbiotic composition can comprise a fermentation process involving the yeast strain in culture. The fermentation process can involve any fermentation step disclosed herein. Following cultivation and / or fermentation of a yeast strain in culture, the number of viable yeast cells can be reduced and / or removed from the culture sample. For instance, viable yeast cells can be removed via pasteurization and / or filtration. In such instances, following the removal of yeast cells, the culture sample can comprise a yeast culture supernatant. The yeast culture supernatant can be a cell-free culture supernatant.

[0280] The postbiotic compositions disclosed herein can be generated by reducing the number of viable yeast cells in a medium (e.g., a cultivation and / or fermentation medium). In some instances, the concentration of yeast cells in a medium is at least about 104CFU / ml to about 1012CFU / ml, about 105CFU / ml to about 1012CFU / ml, about 106CFU / ml to about 1012CFU / ml, about 107CFU / ml to about 1012CFU / ml, about 108CFU / ml to about 1012CFU / ml, about 109CFU / ml to about

[0281] 1012CFU / ml, about 1010CFU / ml to about 1012CFU / ml, about 1011CFU / ml to about 1012CFU / ml, or more than 1012CFU / ml.

[0282] In some instances, the yeast culture is pasteurized. A pasteurization process can reduce the concentration of yeast cells the medium is reduced to no more than about 10-1CFU / ml to about 10-10CFU / ml, about 10-2CFU / ml to about 10-8CFU / ml, about 10-1CFU / ml to about 10-8CFU / ml, 4)

[0283] about 10yCFU / ml to about 10 ' CFU / ml, about 10* CFU / ml to about 106CFU / ml, about 10-' CFU / ml to about 10sCFU / rnl, about 106CFU / ml to about IO4CFU / ml, about 10’ CFU / ml to about 103CFU / ml, about 104CFU / ml to about 102CFU / ml, about 105CFU / ml to about 10 CFU / ml, about 102CFU / ml to about 1 CFU / ml, or less than 1 CFU / ml. In some instances, pasteurization reduces the concentration of yeast m the medium to no more than 1000 CFU / ml, 100 CFU / ml, 10 CFU / ml, or 1 CFU / ml. In some instances, pasteurization reduces the concentration of yeast in the medium to no more than 1000 CFU / ml. In some instances, pasteurization reduces the concentration of yeast in the medium to no more than 100 CFU / ml. In some instances, pasteurization reduces the concentration of yeast in the medium to no more than 10 CFU / ml. In some instances, pasteurization reduces the concentration of yeast in the medium to no more than 1 CFU / ml. In some instances, pasteurization reduces the concentration of yeast in the medium to less than 1 CFU / ml.

[0284] Pasteurization can comprise heating the yeast culture (i.e., medium comprising yeast) to about or at least 90°C. In some embodiments, the yeast culture undergoes high temperature short time pasteurization. In some embodiments, the yeast culture is pasteurized via tube heat exchanger. In some embodiments, the yeast culture is pasteurized via plate heat exchanger. In some embodiments, the yeast culture is pasteurized via spray drying. Pasteurization methods suitable for reducing the number of yeast cells in a culture are known in the art. Any method capable of reducing yeast cells in culture can be used. Pasteurization of a yeast culture does not inactivate and / or degrade PCA present in the yeast culture. That is, PCA present in the yeast culture maintains its activity following pasteurization.

[0285] The number of viable yeast cells in a medium (e.g., a cultivation and / or fermentation medium) can be reduced via filter-sterilization. In such instances, a yeast culture can be subject to a filter sterilization step to remove yeast cells from the culture. Methods of filter-sterilization of yeast cultures are readily understood in the art. Any filter capable of removing yeast from a culture can be used. In some instances, a yeast culture is subject to a 0.22 pM filter to remove and / or reduce the concentration of yeast cells in the filtered sample. Following filter-sterilization, the concentration of yeast cells in a filtered culture sample (i.e., cell-free culture supernatant) is reduced to no more than about

[0286] 1012CFU / rnl to about 10ioCFU / ml, about 1011CFU / ml to about 10yCFU / ml, about 10s0CFU / l to about 10* CFU / ml. about 109CFU / rnl to about IO CFU / ml, about 10sCFU / rnl to about 10° CFU / ml, about 10' CFU / ml to about 10’ CFU / ml, about 10” CFU / ml to about 104CFU / ml, about 105CFU / ml to about 105CFU / ml, about 10* CFU / ml to about I02CFU / ml, about 10' CFU / ml to about 10 CFU / ml, about 10zCFU / ml to about 1 CFU / ml, or less than 1 CFU / ml. In some instances, filter-sterilization reduces the concentration of yeast in the medium to no more than 1000 CFU / ml, 100 CFU / ml, 10 4)

[0287] CFU / ml, or 1 CFU / ml. In some instances, filter-sterilization reduces the concentra tion of yeast in the medium to no more than 1000 CFU / ml. In some instances, filter-sterilization reduces the concentration of yeast in the medium to no more than 100 CFU / ml. In some instances, filter-sterilization reduces the concentration of yeast in the medium to no more than 10 CFU / ml. In some instances, filter-sterilization reduces the concentration of yeast in the medium to no more than 1 CFU / ml. In some instances, filtersterilization reduces the concentration of yeast in the medium to less than 1 CFU / ml.

[0288] Postbiotic compositions disclosed herein comprising a plurality of Saccharomyces cerevisiae var. boulardii, and / or cell constituent(s) thereof, wherein said S. boulardii comprises a heterologous polynucleotide encoding Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or comprises a conservatively substituted version of SEQ ID NO: 1, and wherein 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%, or at least 99% of said plurality is non-viable.

[0289] The postbiotic composition disclosed herein can comprise a plurality of Saccharomyces cerevisiae var. boulardii, and / or cell constituent(s) thereof, wherein said S. boulardii comprises a heterologous polynucleotide encoding Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or comprises a conservatively substituted version of SEQ ID NO: 1, and wherein said plurality comprises no more than: i) 1000 CFU / gram, 100 CFU / gram, 10 CFU / gram, or 1 CFU / gram; and / or ii) 1000 CFU / ml, 100 CFU / ml, 10 CFU / ml, or 1 CFU / ml, of said Saccharomyces cerevisiae var. boulardii.

[0290] The Saccharomyces cerevisiae var. boulardii (S. boulardii)-derived postbiotic compositions can comprise at least one postbiotic agent. In such instances, the postbiotic composition comprises a plurality of Saccharomyces cerevisiae var. boulardii, and / or cell constituent s) thereof, wherein said cell constituent(s) comprises at least one postbiotic agent. 4)

[0291] cell. In some embodiments, the postbiotic agent comprises a secreted microbial protein, metabolite, bioactive compound, components thereof, or combinations thereof.

[0292] Numerous postbiotic agents (e.g., exopolysaccharide, extracellular membrane vesicle(s), genetic material) may be present in the S. boula / 'dii-derived postbiotic compositions (e.g., cell-free S. boulardii-culture supernatant) described herein. Postbiotic agents refer to non-viable agents capable of being produced by microorganisms (e.g., probiotic yeast such as S. boulardii) or byproducts thereof, including compounds secreted by live microorganisms or released after lysis, such as metabolites, enzymes, peptides, teichoic acids, peptidoglycan-derived muropeptides, polysaccharides, oligosaccharides, fatty acids, glycerolipids, purines, sphingolipids, cell surface proteins, or organic acids. For example, the postbiotic may be one or more exopolysaccharide, extracellular membrane vesicle(s), or genetic material. In some aspects, the postbiotic agent comprises genetic material. In some aspects, the genetic material comprises DNA. In some aspects, the genetic material comprises RNA. When the postbiotic comprises RNA, the RNA can include any type of RNA. In some instances, the postbiotic agent comprises multiple forms of RNA. Methods for distinguishing between different forms of RNA are known in the art. In some aspects, the RNA is microRNA. Postbiotics possess different functional properties. These properties can positively affect the microbiota homeostasis and / or the host metabolic and signaling pathways, thus affecting specific physiological, immunological, neuro-hormone, biological, regulatory, or metabolic reactions. A postbiotic agent as described herein can be a byproduct of fermentation. In some instances, a postbiotic agent is present in a microbial cell culture. For instance, a postbiotic agent may be secreted by a microbial cell into a cell culture medium. Such culture medium can be separated from the microbial cells to generate a cell-free culture supernatant. In some instances, the cell culture supernatant is dried (e.g., freeze-dried), as described herein. In some instances, the cell culture supernatant is powderized (e.g., formulated into a powder). A postbiotic agent may be purified from a cell culture supernatant. Additionally, a postbiotic agent as described herein can be a byproduct of microbial killing, such as, for example, killing by pasteurization, sterilization, or spray drying, as described herein.

[0293] In general, postbiotic agents can be differentiated either by their elemental composition, i.e., lipids (e.g. butyrate, propionate, dimethyl acetyl- derived plasmalogen), proteins (e.g. lactocepin, p40 molecule), carbohydrates (e.g. galactose-rich polysaccharides, and teichoic acids), vitamins / co-factors (e.g., B-group vitamins), organic acids (e.g., propionic and 3 -phenyllactic acid) and complexes molecules such as peptidoglycan-derived muropeptides, lipoteichoic acids, or by their physiological 4)

[0294] functions, which include, but are not limited to, immunomodulation, anti-inflammatory, hypocholesterolemic, anti-obesogenic, anti-hypertensive, anti-proliferative, and antioxidant effects.

[0295] In some instances, the postbiotic agent is a short chain fatty acid (SCFA). SCFAs can act as signaling molecules by improving, for example, regulation of lipid metabolism, glucose homeostasis and insulin sensitivity, through the activation of receptors such as G protein-coupled receptors (GPRs), thus contributing in the regulation of energy balance while maintaining metabolic homoeostasis.

[0296] The postbiotic agent can be one or more short chain fatty acids (SCFAs). For example, the postbiotic agent may be acetate, propionate, and / or butyrate, or a derivate, or variant thereof. In some instances, the postbiotic agent is two or more of acetate, propionate, and / or butyrate, or a derivative, or variant thereof. In some instances, the postbiotic agent comprises acetate, propionate, and butyrate, or a derivative, or variant thereof. In some instances, the postbiotic agent comprises lactate, formate, or succinate, or a derivative or variant thereof.

[0297] In particular embodiments, the postbiotic composition can contain a mixture of postbiotic agents. The composition including the postbiotic agent may include any number or type (e.g., classes) of postbiotic agents, such as at least about any one of 1 postbiotic agent, 2, 3, 4, 5, 10, 15, 20, or more postbiotic agents. For example, the postbiotic agent can be a yeast cell lysate containing a heterogeneous mixture of yeast compounds (e.g., cell-free supernatants following lysis of yeast). In some instances, the postbiotic agent can be a cell-free supernatant containing secreted elements from a yeast cell. In other instances, the postbiotic agent can be a purified from a yeast. Alternatively, the postbiotic agent may be synthesized based on known postbiotic agents.

[0298] The postbiotic agent may be formulated in a composition for any of the uses described herein. A suitable concentration of each postbiotic agent in the composition depends on factors such as efficacy, stability of the postbiotic agent, number of distinct postbiotic agents, the formulation, and methods of application of the composition.

[0299] In specific embodiments, a postbiotic agent of the composition disclosed herein is a natural byproduct of the methods disclosed herein. In some embodiments, a postbiotic agent can be added to the postbiotic composition during the production (e.g., fermentation) process. In such instances, a postbiotic agent can be isolated from an external source prior to be added to the postbiotic composition disclosed herein. The postbiotic agent can also be added as part of a whole composition or intact cell during the production process. Postbiotics can be obtained using a variety of methods known in the art. For example, postbiotics can be obtained using cell disruption techniques, which include, but are not limited to, heat (Lee et al., J. of Micro, and Biotech. 12(3):398-405, 2002; Tejada-Simon & Pestka, J. 4)

[0300] of Food Protection. 62(12): 1435-1444, 1999), enzymatic treatments (Li et al., Food Chem. 135:1914-1919, 2012), solvent extraction (Kim et al., Molec. Immun. 48(4):382-391, 2011), or sonication.

[0301] Postbiotic agents can be isolated from a variety of microorganisms (e.g., Gram-negative bacteria, Gram-positive bacteria, or yeast).

[0302] To obtain the postbiotic agent, additional extraction and clean-up steps can be used, such as centrifugation, dialysis, freeze-drying, or column purification. It would be understood that the extraction and clean-up steps remove the postbiotic agent from remaining molecules. In some instances, endonucleases can be used to eliminate nucleic acids, such as DNA and / or RNA from a cell culture supernatant preparation or postbiotic composition. In such instances, an endonuclease would not degrade the postbiotic agent. Alternatively, dialysis can also be used to extract the postbiotic agent. Further, centrifugation can aid in the extraction of postbiotic agents.

[0303] A variety of analytical approaches known in the art can be used for identification of a postbiotic agent. The selection of instrumental technique depends on the analytical goals and the type of characterization (qualitative and / or quantitative) pursued. For example, mass spectrometry (e.g., matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, Fourier transform ion cyclotron resonance mass spectrometry, or electro-spray ionization mass spectrometry), High Performance Liquid Chromatography (HPLC), Ultra Performance Liquid Chromatography (UPLC) chromatography coupled with tandem mass spectrometry, or spectroscopy (e.g., proton nuclear magnetic resonance spectroscopy (1H-NMR, C-13 NMR) can be used to identify or validate a postbiotic agent for use in accordance with the present methods.

[0304] Disclosed herein are Saccharomyces cerevisiae var. boulardii (S. boulardii) -der ved postbiotic compositions comprising a plurality of S. boulardii, and / or cell constituent(s) thereof, wherein said plurality of S. boulardii are capable of producing PCA. As such, postbiotic compositions of the present disclosure can further comprise PCA. PCA can be present at any concentration. In some instances, the postbiotic composition comprises an effective amount of PCA. An “effective amount” when used in the context of PCA refers to any quantity of PCA which achieves a desired result of any method described herein. In particular, an effective amount of PCA can maintain and / or alter at least one biological activity in a subject following administration of a postbiotic composition.

[0305] In some instances, PCA is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the postbiotic composition weight per volume (w / v), or volume per volume (v / v). In some instances, PCA 4)

[0306] is present at a concentration of at least 0.0001 mM (i.e., 0.1 pM), at least 0.0005 mM (i.e., 0.5 pM), 0.001 mM (i.e., 1 pM), at least 0.005 mM, at least 0.01 mM, at least 0.03 mM, at least 0.06 mM, at least 0.12 mM, at least 0.25 mM, at least 0.5 mM, at least 1 mM, at least 2 mM, at least 5 mM, or more than 5 mM. In certain instances, PCA is present at a concentration of between about 0.5 pM to about 5 pM (i.e, 0.005 mM), about 1 pM to about 10 pM (i.e., 0.01 mM), about 0.005 mM to about 0.03 mM, about 0.01 mM to about 0.06 mM, about 0.03 mM to about 0.12 mM, or about 0.06 mM to about 0.25 mM.

[0307] PCA can be present in the Saccharomyces cerevisiae var. boulardii (S. boulardii-derived postbiotic compositions at a concentration of between about 0.001 mM to about 0.01 mM, about 0.002 mM to about 0.01 mM, about 0.003 mM to about 0.01 mM, about 0.004 mM to about 0.01 mM, about 0.005 mM to about 0.01 mM, about 0.006 mM to about 0.01 mM, about 0.007 mM to about 0.01 mM, about 0.008 mM to about 0.01 mM, or about 0.009 mM to about 0.01 mM. In other instances, PCA is present at a concentration of between about 0.01 mM to about 0.03 mM, about 0.011 mM to about 0.03 mM, about 0.012 mM to about 0.03 mM, about 0.013 mM to about 0.03 mM, about 0.014 mM to about 0.03 mM, about 0.015 mM to about 0.03 mM, about 0.016 mM to about 0.03 mM, about 0.017 mM to about 0.03 mM, about 0.018 mM to about 0.03 mM, about 0.019 mM to about 0.03 mM, about 0.02 mM to about 0.03 mM, about 0.022 mM to about 0.03 mM, about 0.023 mM to about 0.03 mM, about 0.024 mM to about 0.03 mM, about 0.025 mM to about 0.03 mM, about 0.026 mM to about 0.03 mM, about 0.027 mM to about 0.03 mM, about 0.028 mM to about 0.03 mM, about 0.029 mM to about 0.03 mM.

[0308] In some instances, PCA is present at a concentration of about 0.12 mM. In some instances, PCA is present at a concentration of about 0.03 mM. In some instances, PCA is present at a concentration of about 0.01 mM. In some instances, PCA is present at a concentration of about 0.005 mM.

[0309] The S. boulardii-& Q we& postibiotic compositions disclosed herein may comprise PCA at any concentration disclosed herein. In some instances, the postbiotic composition is a S. boulardii cell culture supernatant, e.g., a cell-free culture supernatant. In some instances, the postbiotic composition is a S. boulardii fermentation culture supernatant. In some instances, the postbiotic composition comprises a formulated postbiotic composition. In such instances, the postbiotic composition comprises a food and / or beverage product. Such postbiotic compositions can comprise between 2 g / L to 4 g / L, between 3 g / L to 5 g / L, between 4 g / L to 6 g / L, between 5 g / L to 7 g / L, between 6 g / L to 8 g / L, between 7 g / L to 9 g / L, between 8 g / L to 10 g / L, between 9 g / L to 11 g / L, between 10 g / L to 12 4)

[0310] g / L, between 11 g / L to 13 g / L, between 12 g / L, to 14 g / L, between 13 g / L to 15 g / L, between 14 g / L to 16 g / L. between 15 g / L to 17 g / L, between 16 g / L to 18 g / L, between 17 g / L to 19 g / L, between 18 g / L to 20 g / L, between 19 g / L to 21 g / L. between 20 g / L to 22 g / L, between 21 g / L to 23 g / L. between 22 g / L to 24 g / L, between 23 g / L to 25 g / L, between 24 g / L to 26 g / L. between 25 g / L to 27 g / L, between 26 g / L to 28 g / L, between 27 g / L to 29 g / L, between 28 g / L to 30 g / L, between 29 g / L to 31 g / L, between 30 g / L to 32 g / L, between 31 g / L to 33 g / L, between 32 g / L to 34 g / L, between 33 g / L to 35 g / L, between 34 g / L to 36 g / L, between 35 g / L to 37 g / L, between 36 g / L to 38 g / L, between 37 g / L to 39 g / L, between 38 g / L to 40 g / L, between 39 g / L to 41 g / L, between 40 g / L to 42 g / L, between 41 g / L to 43 g / L, between 42 g / L to 44 g / L. between 43 g / L to 45 g / L, between 44 g / L to 46 g / L, between 45 g / L to 47 g / L, between 46 g / L to 48 g / L, between 47 g / L to 49 g / L, between 48 g / L to 50 g / L, between 49 g / L to 51 g / L, between 50 g / L to 52 g / L, between 51 g / L to 53 g / L, between 52 g / L to 54 g / L, between 53 g / L to 55 g / L, between 54 g / L to 56 g / L, between 55 g / L to 57 g / L, between 56 g / L to 58 g / L, between 57 g / L to 59 g / L, between 58 g / L to 60 g / L, or more than 60 g / L of PCA. In some embodiments, the postbiotic composition comprises a liquid composition (e.g., a cell culture supernatant or beverage product). In some embodiments, the postbiotic composition comprises a solid composition (e.g., a lyophilized; freeze-dried; and / or powdered composition). In some instances, the postbiotic composition comprising 5. boulardii and / or cell constituents) thereof comprises PCA at a concentration of at least 2 g / L, at least 4 g / L, at least 6 g / L, at least 8 g / L, at least 10 g / L, at least 12 g / L, at least 14 g / L, at least 16 g / L, at least 18 g / L, at least 20 g / L, at least 22 g / L, at least 24 g / L, at least 26 g / L, at least 28 g / L, at least 30 g / L, at least 32 g / L, at least 34 g / L, at least 36 g / L, at least 38 g / L, at least 40 g / L, at least 42 g / L, at least 44 g / L, at least 46 g / L, at least 48 g / L, at least 50 g / L, at least 52 g / L, at least 54 g / L, at least 56 g / L, at least 58 g / L. at least 60 g / L.

[0311] In some instances, PCA is present in a A bo« / rt / zz-derived postbiotic composition at any concentration depicted in Tables 9-11.

[0312] Disclosed herein are Saccharomyces cerevisiae var. boulardii (S. boulardii) -derived postbiotic composition, said composition comprising a plurality of S. boulardii, and / or cell constituent(s) thereof, wherein said S. boulardii comprises a heterologous polynucleotide encoding a Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ,

[0313] wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1, and 4)

[0314] wherein 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%, or at least 99% of said plurality is non-viable. The AroZ or the variant of AroZ can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of SEQ ID NO: 1. In some instances, the variant comprises 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% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The Saccharomyces cerevisiae can comprise a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1.

[0315] The S. boulardii can comprise any additional heterologous polynucleotide disclosed herein. For instance, the S. boulardii can comprise one or more heterologous polynucleotides, wherein said one or more heterologous polynucleotides comprise a heterologous polynucleotide sequence encoding any of the following genes: arol, aro3, aro4, tkll, and tall.

[0316] In some instances, the S. boulardii-derived postbiotic composition comprises a plurality of S. boulardii and / or cell constituent(s) thereof, wherein said S. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, wherein said S. boulardii further comprises the following heterologous polynucleotides encoding codon-optimized variants: i) a second heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkll enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; and vii) an eighth heterologous polynucleotide encoding a Tall enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7.

[0317] The postbiotic compositions disclosed herein can be administered to any tissue of a subject. As such, the postbiotic compositions can further comprise cells and / or tissue of said subject. In particular, the postbiotic compositions disclosed herein can further comprise i) luminal contents of the 4)

[0318] gastrointestinal tract of a subject; ii) at least one intestinal epithelial cell of a subject; or iii) or at least one skin epithelial cell of a subject.

[0319] Methods

[0320] Disclosed here are methods for altering or maintaining at least one biological activity in a subject. Such methods can be performed in vivo in a subject and involve administering to a subject an effective amount of the microbial compositions, the postbiotic compositions, and / or the food or beverage products disclosed herein. The disclosed Saccharomyces cerevisiae var. boulardii comprising a heterologous polynucleotide encoding an AroZ can be used to alter or maintain at least one biological activity in a subject. The biological activity can include, but is not limited to, i) suppressing expression of at least one gene associated with inflammation in at least one cell present in said subject; ii) stimulating an epithelial tissue response in said subject; iii) reducing at least one symptom of a gastrointestinal or skin disorder in said subject; and / or iv) improving well-being of said subject, compared to a control, wherein said control comprises a subject who is not administered an effective amount of said composition. A control will be understood to be dependent upon the specific biological activity being measured. Proper controls are described throughout the present application. Any control can be utilized when assessing the disclosed methods.

[0321] Also provided herein are methods of improving the antioxidant status of a subject. Antioxidant status refers to the balance of antioxidants in the body, which play a crucial role in protecting cells from oxidative stress and maintaining overall health.

[0322] Also provided herein are methods of decreasing inflammation in a subject. In some instances, inflammation is decreased in a tissue of a subject. Such methods comprise administering an effective amount of any microbial composition or postbiotic composition disclosed herein.

[0323] Any of the methods disclosed herein can comprise administration of at least one host cell, wherein said host cell comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1. In some instances, the host cell is a Saccharomyces. In such instances, the host cell can be a Saccharomyces cerevisiae cell or a Saccharomyces cerevisiae var. boulardii cell. 4)

[0324] Any of the methods disclosed herein can comprise administration of at least one 5. boulardii and / or cell constituent(s) thereof, wherein said S. boulardii host cell comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, wherein said S. boulardii further comprises the following heterologous polynucleotides encoding codon-optimized variants: i) a second heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkll enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; and vii) an eighth heterologous polynucleotide encoding a Tall enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7.

[0325] Any of the methods disclosed herein can comprise administration of a microbial composition comprising a plurality of S. boulardii and / or cell constituent(s) thereof, wherein said S. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, wherein said S. boulardii further comprises the following heterologous polynucleotides encoding codon-optimized variants: i) a second heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkll enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; and vii) an eighth heterologous polynucleotide encoding a Tall enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7.

[0326] Any of the methods disclosed herein can comprise administration of a S. boulardii-den\ed postbiotic composition comprising a plurality of S. boulardii and / or cell constituent(s) thereof, wherein 4)

[0327] said 5. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1, wherein said S. boulardii further comprises the following heterologous polynucleotides encoding codon- optimized variants: i) a second heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3; ii) a third heterologous polynucleotide encoding a second Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11; iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4; iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12; v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5; vi) a seventh heterologous polynucleotide encoding a Tkll enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; and vii) an eighth heterologous polynucleotide encoding a Tall enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7.

[0328] In some embodiments, administration of an engineered S. boulardii and / or cell constituent(s) thereof described herein can reduce or treat an inflammatory disease, and particularly can ameliorate the symptoms or prevent an inflammatory disease of the gastrointestinal tract, such as an inflammatory bowel disease (IBD), including, but not limited to, Crohn’s disease and / or colitis (e.g., ulcerative colitis), in a subject. In some embodiments, administration of engineered S. boulardii and / or cell constituent(s) thereof described herein can reduce or treat an inflammatory disease, and particularly can ameliorate the symptoms or prevent a systemic inflammatory disorder outside the gastrointestinal tract, such as rheumatoid arthritis, systemic lupus erythematosus and / or multiple sclerosis, or an inflammatory skin disorder, in a subject. “Treatment” is herein defined as curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the condition or the symptoms of a subject with a gastrointestinal disorder or systemic inflammatory disorder. The systemic inflammatory disorder can be any inflammatory disorder and need not be associated with gastrointestinal inflammation. The subject to be treated can be suffering from or at risk of developing a gastrointestinal disorder, including, for example, be suffering from an inflammatory bowel disease or be at risk of developing an inflammatory bowel disease. Administration of the engineered S. boulardii and / or cell constituent(s) thereof, or composition comprising said engineered S. boulardii and / or cell constituent(s) thereof described herein, can be for either a prophylactic or therapeutic purpose. By “preventing” is intended that the yeast is provided prophylactically, i.e., the yeast is provided in advance of any symptom. The prophylactic administration of the yeast strain composition described 4)

[0329] herein serves to prevent or attenuate any subsequent symptom. When provided therapeutically, the yeast strain composition is provided at (or shortly after) the onset of a symptom. The therapeutic administration of the substance may serve to attenuate any actual symptom. In some embodiments, the reduction or decrease in inflammation may include stimulation of intestinal integrity; reduction of intestinal permeability; improvement of mucin synthesis, secretion, and / or quality; improvement of the maturation and differentiation of the intestinal epithelium; improvement of nutrient absorption; increase of the production of soluble factors that transfer antimicrobial activity; stimulation of, improvement of, or support of resistance to infection; support of cellular or humoral responses against viral or bacterial infection; increased cytotoxicity (both anti-viral and anti-tumor); support of systemic and / or mucosal vaccination responses; increase or support of cellular and / or humoral immunity; increase or support of natural immunity (including neutrophils, phagocytes, macrophages, and natural killer cell activity); increase or support of adaptive T and B cell immunity; stimulation of a helper T cell 1 (Thl) cytokine pattern (increased IL-1, IL-2, IFN-gamma, IL- 12, TNF-alpha; human leukocyte antigen-Dr (HLA-Dr) expression); suppression of inflammation or production of systemic and mucosal inflammatory mediators (including cytokines and / or chemokines); reduction of sensitization by reducing total and / or allergen- specific IgE; reduction of the production of allergic cytokines; reduction of a Th2 supporting immunoglobulin profile; and combinations thereof. As used herein, the term “antiinflammatory cytokine” refers to a naturally occurring or recombinant protein, analog thereof or fragment thereof that elicits an anti-inflammatory response in a cell that has a receptor for that cytokine. Anti-inflammatory cytokines of the invention can be immunoregulatory molecules that control the proinflammatory cytokine response. Anti-inflammatory cytokines of the invention include interleukin (IL)-l receptor antagonist, IL-4, IL-10, IL-11, and IL-13, IL-16, IFN-alpha, TGF-beta, G-CSF. As used herein, the term “proinflammatory cytokine” refers to an immunoregulatory cytokine that favors inflammation. Proinflammatory cytokines of the invention include IL1- alpha, ILl-beta, TNF-alpha, IL-2, IL-3, IL-6, IL-7, IL-9, IL- 12, IL- 17, IL- 18, LT, LIF, Oncostatin, or IFN-alpha, IFN-beta, IFN-gamma. In some embodiments, administration of the bacterial strain composition results in an increase in anti-inflammatory cytokine production. As used herein, an “increase in” or “increasing” anti-inflammatory cytokine production comprises any statistically significant increase the antiinflammatory cytokine level when compared to an appropriate control. Such increases can include, for example, at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or greater increase in the anti-inflammatory cytokine level. Such increases can also include, for example, at least about a 3%-l 5%, 10%-25%, 20% to 35%, 30% to 45%, 40%-55%, 50%-65%, 60%-75%, 70%- 4)

[0330] 85%, 80%-95%, 90%- 105%, 100%-115%, 105%-120%, 115% -130%, 125%-150%, 140%-160%, 155%-500% or greater increase in the anti-inflammatory cytokine level. Methods to assay for the level of anti-inflammatory cytokine level, are known. See, for example, Leng S., et al. (2008) J Gerontol A Biol Sci Med Sci 63(8): 879-884. Methods to assay for the production of anti- inflammatory cytokines include multiplex bead assay, ELISA, ELISPOT, qPCR, and flow cytometry. See, for example, Maecker et al. (2005) BMC Immunology 6: 13. Methods and compositions also include those which decrease proinflammatory cytokine production, which may decrease or prevent an inflammatory response. As used herein, a decrease in the level of pro-inflammatory cytokine production comprises any statistically significant decrease in the level of pro-inflammatory cytokine production in a subject when compared to an appropriate control. Such decreases can include, for example, at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in the level of proinflammatory cytokines. Methods to assay for cytokine levels are known and include, for example Leng S., et al. (2008) J Gerontol A Biol Sci Med Sci 63(8): 879-884. Methods to assay for the production of pro-inflammatory cytokines include multiplex bead assay, ELISPOT and flow cytometry. See, for example, Maecker et al. (2005) BMC Immunology 6:13. Inflammatory cytokine production can also be measured by assaying the ratio of anti- inflammatory cytokine production to proinflammatory cytokine production. In specific aspects, the ratio of anti-inflammatory cytokine production to proinflammatory cytokine production is increased by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 300, 600, 900, 1000 fold or greater when compared to an appropriate control. In other aspects, the ratio of anti-inflammatory cytokine production to pro-inflammatory cytokine production is increased by about 1 to 5 fold, about 5 to 10 fold, about 10 to 20 fold, about 20 to 30 fold, about 30 to 40 fold, about 40 fold to 60 fold, about 60 fold to 80 fold, about 80 fold to about 100 fold, about 100 to 200 fold, about 200 fold to 300 fold, about 300 to 400 fold, about 400 to about 500 fold, about 500 to about 600 fold, about 600 fold to about 700 fold, about 700 fold to 800 fold, about 800 fold to about 1000 fold or greater when compared to an appropriate control. Methods to determine the ratio of anti-inflammatory cytokine production to pro- inflammatory cytokine production can be found, for example, Leng S., et al. (2008) J Gerontol A Biol Sci Med Sci 63(8): 879-884. Methods to assay for the production of cytokines include multiplex bead assay, ELISA, ELISPOT, qPCR, and flow cytometry. See, for example, Maecker et al. (2005) BMC Immunology 6:13. In specific embodiments, administration of an effective amount of the engineered S. boulardii and / or cell constituent(s) thereof described herein, or composition comprising the engineered S. boulardii and / or cell constituent(s) thereof described herein, can decrease the expression of a marker of inflammation compared to a proper control. Such decreases 4)

[0331] can include, for example, at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in the level of expression of a marker of inflammation, as measured by mRNA level, qPCR, protein level, ELISA, LPS levels, or any method known in the art. In specific embodiments, the marker of inflammation comprises Cox-2, iNOS, and / or IL-6. The level of the inflammation marker can be measured as it relates to a proper control. As used herein a proper control includes but is not limited to, the expression level of a marker of inflammation in a corresponding sample from a subject that was not administered the yeast strain and / or cell constituents thereof, the expression level of a marker of inflammation in a sample from a subject prior to administration of the yeast strain and / or cell constituents thereof, or the expression level of a marker of inflammation in a standardized sample from a subject that was not administered the yeast strain and / or cell constituents thereof. One of skill in the art would be able to identify proper controls in order to measure an increase in the expression level of a marker of inflammation in a human subject or agricultural animal.

[0332] For instance, administration of a composition comprising the disclosed Saccharomyces cerevisiae var. boulardii, and / or cell constituents thereof, can be used to reduce (e.g., suppress) inflammatory gene expression in host cells. Expression of any gene associated with inflammation in a host cell is envisaged as capable of being reduced by the disclosed compositions. In some instances, expression of a gene associated with inflammation is similar following administration of the disclosed compositions as a control that is not exposed to an inflammatory signal in the host. A Saccharomyces cerevisiae var. boulardii-derived postbiotic composition may reduce gene expression of a cell that comes into contact with at least one proinflammatory host molecule in a subject after being contacted with said postbiotic composition. In such instances, it would be understood that the cell exhibiting reduced expression may exhibit expression of said proinflammatory gene at levels that are elevated compared to the same cell type which is not exposed to at least one proinflammatory molecule.

[0333] Nevertheless, said cell exhibits reduced gene expression compared to the same cell type that is contacted with at least one proinflammatory molecule in the absence of contact with the postbiotic composition. The level of gene expression modulation (i.e., reduction) would be understood to be dependent upon time period after which a cell in a subject comes into contact with a proinflammatory molecule. In some instances, gene expression is suppressed within 60 minutes, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, within 19 hours, within 20 hours, 4)

[0334] within 21 hours, within 22 hours, within 23 hours, within 24 hours, or greater than 24 hours following administration of the postbiotic composition.

[0335] Administration of compositions comprising Saccharomyces cerevisiae var. boulardii comprising a heterologous polynucleotide encoding an AroZ and / or cell constituents thereof (e.g., a Saccharomyces cerevisiae var. boulardii-derived postbiotic composition described herein) can reduce gene expression of iNOS in a cell in a subject. The host cell can be a mammalian cell comprising an iNOS gene. Any mammalian cell comprising an iNOS gene is envisaged as exhibiting reduced iNOS expression. In some instances, the mammalian cell is a cell in the gastrointestinal tract of a subject. In another instance, the mammalian cell is an epithelial cell. Expression of iNOS can be reduced at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to a control cell. A control cell can include the same mammalian cell type in the same tissue which is not contacted with the composition comprising S. cerevisiae and / or cell constituents thereof. One of skill would understand that expression is reduced compared to a control cell.

[0336] Expression of iNOS can be reduced by about 50% following administration of a Saccharomyces cerevisiae var. boulardii-derived postbiotic composition, comprising a plurality of Saccharomyces cerevisiae var. boulardii, and / or cell constituent(s) thereof. In some instances, expression of iNOS can be reduced between 45% to 55%. In another instance, expression of COX-2 can be reduced about 45 to 47%, about 46% to 48%, about 47 to 49%, about 48 to about 50%, about 49% to about 51%, about 50% to about 52%, about 51% to about 53%, about 52% to about 54%, or about 53% to about 55%. Reduced iNOS expression can be dependent upon the period of time after which the mammalian cell comes into contact with a proinflammatory signal.

[0337] iNOS gene expression can be reduced within at least 1 hour, at least 2 hours, at least 3 hours at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, or more than 24 hours after a mammalian host cell is contacted with at least one proinflammatory molecule. For instance, iNOS expression in a mammalian cell may be reduced when measured at least 8 hours after contact with a proinflammatory signal. In such instances, iNOS expression may be reduced to a greater extent compared to a reduction seen when measured after 4 hours. 4)

[0338] In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% within 6-10 hours after said mammalian cell is contacted with at least one proinflammatory molecule.

[0339] In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% within 7-9 hours after said mammalian cell is contacted with at least one proinflammatory molecule.

[0340] In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% within 5 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% within 6 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% within 7 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% within 8 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% within 9 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% within 10 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% within 11 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% within 12 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% more than 12 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of iNOS in a mammalian cell can be reduced between 45% to 55% less than 24 hours after said mammalian cell is contacted with at least one proinflammatory molecule.

[0341] Administration of compositions comprising Saccharomyces cerevisiae var. boulardii comprising a heterologous polynucleotide encoding an AroZ and / or cell constituents thereof (e.g., a Saccharomyces cerevisiae var. boulardii-derived postbiotic composition described herein) can reduce gene expression of Cox-2 in a cell in a subject. The host cell can be a mammalian cell comprising a Cox-2 gene. Any mammalian cell comprising an iNOS gene is envisaged as exhibiting reduced Cox-2 4)

[0342] expression. In some instances, the mammalian cell is a cell in the gastrointestinal tract of a subject. In another instance, the mammalian cell is an epithelial cell. Expression of Cox-2 can be reduced at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to a control cell. A control cell can include the same mammalian cell type in the same tissue which is not contacted with the composition comprising S. cerevisiae and / or cell constituents thereof. One of skill would understand that expression is reduced compared to a control cell.

[0343] Expression of Cox-2 can be reduced by about 50% following administration of a Saccharomyces cerevisiae var. boulardii-derived postbiotic composition, comprising a plurality of Saccharomyces cerevisiae var. boulardii, and / or cell constituent(s) thereof.

[0344] In some instances, expression of Cox-2 can be reduced between 45% to 55%. In another instance, expression of Cox-2 can be reduced about 45 to 47%, about 46% to 48%, about 47 to 49%, about 48 to about 50%, about 49% to about 51%, about 50% to about 52%, about 51% to about 53%, about 52% to about 54%, or about 53% to about 55%. Reduced Cox-2 expression can be dependent upon the period of time after which the mammalian cell comes into contact with a proinflammatory signal.

[0345] Cox-2 gene expression can be reduced within at least 1 hour, at least 2 hours, at least 3 hours at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, or more than 24 hours after a mammalian host cell is contacted with at least one proinflammatory molecule. For instance, Cox-2 expression in a mammalian cell may be reduced when measured at least 4 hours after contact with a proinflammatory signal. In some instances, Cox-2 expression in a mammalian cell may be reduced when measured at least 8 hours after contact with a proinflammatory signal.

[0346] In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 1-24 hours after said mammalian cell is contacted with at least one proinflammatory molecule.

[0347] In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 2-10 hours after said mammalian cell is contacted with at least one proinflammatory molecule.

[0348] In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 1 hour after said mammalian cell is contacted with at least one proinflammatory molecule. 4)

[0349] In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 2 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 3 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 4 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 5 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 6 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 7 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 8 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 9 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 10 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 11 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% within 12 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% more than 12 hours after said mammalian cell is contacted with at least one proinflammatory molecule. In some instances, expression of Cox-2 in a mammalian cell can be reduced between 45% to 55% less than 24 hours after said mammalian cell is contacted with at least one proinflammatory molecule.

[0350] Administration of the compositions comprising can suppress gene expression of COX-2 and iNOS in a host cell by any levels described herein.

[0351] Also disclosed herein are methods for stimulating cell migration in a subject. Cell migration can be increased in a tissue of a subject, such as an epithelial tissue. In such instances, the epithelial cell migration would be increased. Migration of cells in any tissue for which the disclosed compositions comprising PCA is administered. In such instances, a composition comprising PCA at 4)

[0352] any concentration disclosed herein can be administered to a tissue of a subject. In one particular embodiment, the tissue can be skin. Any formulation for delivery of a composition disclosed herein comprising PCA is envisaged, e.g., a hydrogel, softgel, lotion, or cream. The use of PCA compositions for enhancing would healing has been demonstrated in the art (Zhou et al., Protocatechuic acid-mediated injectable antioxidant hydrogels facilitate wound healing, Composites Part B: Engineering, Volume 250, 2023, 110451). It would be understood that any of probiotic and / or postbiotic compositions disclosed herein could be used to enhance would healing and / or cell migration compared to a control, wherein said control involves a subject that is not administered the composition.

[0353] Also disclosed herein are methods for stimulating an epithelial tissue response in a subject. The epithelial tissue response can be in any tissue of the subject. For instance, an epithelial tissue response can be stimulated in the gastrointestinal tract or skin of a subject.

[0354] The methods of stimulating an epithelial tissue response can comprise administering to a subject an effective amount of a microbial composition. Any microbial composition of the present disclosure can be administered. In some instances, a microbial composition comprising an engineered S. boulardii described herein is administered. In such instances, the microbial composition can comprise a plurality of S. boulardii, and / or cell constituent(s) thereof, comprising a heterologous polynucleotide encoding Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or comprises a conservatively substituted version of SEQ ID NO: 1, and wherein said plurality comprises about 104CFU / gram to about 1012CFU / gram or about 104CFU / ml to about 1012CFU / ml of. boulardii. The microbial composition is administered at any effective amount. An “effective amount” when used in the context of a microbial composition refers to a quantity of a microbial composition capable of stimulating an epithelial tissue response.

[0355] The methods of stimulating an epithelial tissue response can comprise administering to a subject an effective amount of a postbiotic composition. Any postbiotic composition of the present disclosure can be administered. In some instances, a S. boulardii-derived postbiotic composition is administered. In such instances, the S. boulardii-derived postbiotic composition can comprise a plurality of S. boulardii, and / or cell constituent(s) thereof, comprising a heterologous polynucleotide encoding a Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or comprises a conservatively 4)

[0356] substituted version of SEQ ID NO: 1, and wherein 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%, or at least 99% of said plurality is non-viable. In some instances, said plurality comprises no more than: i) 1000 CFU / gram, 100 CFU / gram, 10 CFU / gram, or 1 CFU / gram; and / or h) 1000 CFU / ml, 100 CFU / ml, 10 CFU / ml, or 1 CFU / ml, of S. boulardii. The postbiotic composition is administered at any effective amount. An “effective amount” when used in the context of a postbiotic composition refers to a quantity of a postbiotic composition capable of stimulating an epithelial tissue response.

[0357] The methods of stimulating an epithelial tissue response in a subject can comprise administering any food or beverage product disclosed herein. The food or beverage products are expected to be as effective as the microbial composition or postbiotic compositions they comprise. A food and / or beverage product can be formulated to include an effective amount of a postbiotic composition.

[0358] The epithelial tissue response can be characterized by at least one epithelial cell within said epithelial tissue exhibiting a cellular response. A cellular response can be any cellular response that contributes to a desired outcome described herein. For example, the cellular response can involve increased production and / or secretion of, or increased expression of a gene encoding, at least one molecule in at least one epithelial cell; and / or increased restitution (e.g., wound closure). The cellular response can include a change in gene expression and / or production and / or secretion of a protein or cellular molecule or metabolite. In some instances, the cellular response includes a change in expression of at least one gene in at least one epithelial cell. The gene can be any epithelial gene associated with epithelial restitution or epithelial wound healing. In some instances, the cellular response includes a change in multiple genes in at least one epithelial cell. It is likely that a response would be seen in more than one epithelial cell. The cellular response can include a change in gene expression and / or production and / or secretion of a protein or cellular molecule or metabolite in at least one epithelial cell. The cellular response can involve such a response in at least 1, at least 10, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010, at least 1011, at least 1012, at least 1013, at least 1014, at least 1015, at least 1016, at least 1017, at least 1018, at least 1019, at least IO20, or more than 1020epithelial cells.

[0359] At least one epithelial cell can exhibit increased production and / or secretion of, or increased expression of a gene encoding, at least one molecule compared to a control cell or tissue. At least 1, at least 10, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010, at least 1011, at least 1012, at least 1013, at least 1014, at least 1015, at least 1016, at least 4)

[0360] 1017, at least 1018, at least 1019, at least 1020, or more than 1020epithelial cells can exhibit increased production and / or secretion of, or increased expression of a gene encoding, at least one molecule compared to a control cell and / or tissue. A control cell can include an epithelial cell in the same tissue of a subject that was not administered the postbiotic composition. A control cell can also include an epithelial cell in the same tissue of the subject prior to, or immediately after, administration of the postbiotic composition. By “immediately after” in reference to administration of the postbiotic composition means within 5 minutes, within 10 minutes, within 15 minutes, within 20 minutes, within 25 minutes, within 30 minutes, within 35 minutes, within 40 minutes, within 45 minutes, within 50 minutes, within 55 minutes, within 60 minutes, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, or within 24 hours following administration of the postbiotic composition. A control tissue can include a plurality of epithelial cells, such as an epithelial monolayer. Methods of examining gene expression, and / or protein production and / or secretion from an epithelial cell and / or tissue would be readily available to a skilled artisan. Any method known can be used. Any method of culturing epithelial cells and / or epithelial cell monolayers (i.e., epithelial tissue) and examining increased expression and / or production and / or secretion of proteins can be used. For instance, gene expression in at least one epithelial cell, and or a monolayer of epithelial cells, can be examined using genomic sequence quantification methods, such as polymerase chain reaction (e.g., qPCR, RT-PCR). Gene expression can be measured by quantifying RNA (e.g., mRNA). The epithelial tissue can comprise at least one epithelial cell. In some instances, the epithelial tissue comprises at least 10, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010, at least 1011, at least 1012, at least 1013, at least 1014, at least 1015, at least 1016, at least 1017, at least 1018, at least 1019, at least 1020, or more than 1020epithelial cells. Importantly, the control cell and / or tissue can be exposed to at least one pro- inflammatory molecule. The postbiotic compositions are particularly effective at enhancing gene expression, and / or molecule production and / or secretion, of a molecule that is increased in the presence of at least one pro-inflammatory molecule. Pro-inflammatory molecules would be readily known by a skilled artisan.

[0361] Administration of an effective amount of a microbial composition or postbiotic composition described herein can increase restitution of epithelial tissue in a subject compared to a control epithelial tissue. Restitution can be measured by monitoring epithelial tissue (e.g., an epithelial monolayer) for signs of restitution and / or wound healing. The epithelial tissue can be damaged prior to restitution monitoring. Damage can occur through any means that can damage an epithelial monolayer. Restitution and / or wound healing can be monitored visually, such as through microscopy. For 4)

[0362] example, the abrasion or wound of an epithelial monolayer can be monitored for reduction in size to monitor wound healing. Any method for monitoring wound healing and / or epithelial restitution can be used. Epithelial restitution can be compared to a control. A control can include an epithelial tissue that receives no treatment following injury. A control can include an epithelial tissue wherein the postbiotic composition disclosed herein is not added to the epithelial tissue following injury. The control can include an epithelial tissue that is treated with something that is not the postbiotic composition disclosed herein.

[0363] Epithelial restitution can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95%. In some instances, epithelial restitution can be increased by 10%-20%, 15%-25%, 20%-30%, 25%-35%, 30%-40%, 35%-45%, 40%-50%, 45%-55%, 50%-60%, 55%-65%, 60%-70%, 65%-75%, 70%-80%, 75%-85%, 80%-90%, or greater than 90%. In another aspect, restitution can be increased at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold.

[0364] In a particular instance, epithelial restitution is increased between 10-15%. Epithelial restitution can be increased at least 10%, at least 11%, at least 12%, at least 13%, or at least 15%. In some instances, epithelial restitution is increased between 10-12%, between 11-13%, between 12-14%, or between 13-15%, compared to a control.

[0365] Administration of a microbial composition or postbiotic composition disclosed herein can increase wound healing (i.e., wound closure) of an epithelial monolayer compared to a control. Wound closure can be measured using any method known to one having skill in the art. For instance, would closure can be measured using microscopy and imaging the epithelial cell monolayer. In such instances, wound closure can be visualized and / or the wound (e.g., the size of the wound) can be measured. A control can include an epithelial tissue wherein the postbiotic composition disclosed herein is not added to the epithelial tissue following injury. The control can include an epithelial tissue that is treated with something that is not the postbiotic composition disclosed herein. Following administration of the postbiotic composition disclosed herein, wound healing and / or closure can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Following administration of a postbiotic composition, wound healing and / or 4)

[0366] closure can be at least 10%-20%, 15%-25%, 20%-30%, 25%-35%, 30%-40%, 35%-45%, 40%-50%, 45%-55%, 50%-60%, 55%-65%, 60%-70%, 65%-75%, 70%-80%, 75%-85%, 80%-90%, or greater than 90%. In some instances, would closure and / or healing can be greater than 80%. Following administration of the postbiotic composition, would healing and / or closure can be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 than 99%. Following administration of the postbiotic composition, would healing and / or closure can be 80%-82%, 81%-83%, 82%-84%, 83%-85%, 84%-86%, 85%-87%, 86%-88%, 87%-89%, 88%-90%, 89%-91%, 90%-92%, 91%-93%, 92%-94%, 93%-95%, 94%-96%, 95%-97%, 96%-98%, 97%-99%, or greater than 99%. Following administration of the postbiotic composition, the percent wound healing and / or closure of the epithelial barrier is greater than epithelial barrier would healing and / or closure percentage in the absence of the postbiotic composition. In such instances, the percent wound healing and / or closure in the absence of a postbiotic is no more than 50%, and percent wound healing and / or closure following administration of the postbiotic composition is greater than 50%.

[0367] Following administration of a microbial composition or postbiotic composition disclosed herein, wound healing and / or closure can be increased at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Following administration of a postbiotic composition, wound healing and / or closure can be at least 10%-20%, 15%-25%, 20%-30%, 25%-35%, 30%-40%, 35%-45%, 40%-50%, 45%-55%, 50%-60%, 55%-65%, 60%-70%, 65%-75%, 70%-80%, 75%-85%, 80%-90%, or greater than 90%. Following administration of the postbiotic composition, wound healing and / or closure can be increased at least 1.1 -fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 14-fold, at least 15 -fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold. Following administration of the postbiotic composition, wound healing and / or closure can be increased 1.4-fold to 1.6-fold, 1.5-fold to 1.7-fold, 1.6-fold to 1.8-fold, 1.7-fold to 1.9-fold, or 1.8-fold to 2-fold.

[0368] In a particular instance, would healing and / or closure is increased between 10-15%. Epithelial restitution can be increased at least 10%, at least 11%, at least 12%, at least 13%, or at least 15%. In 4)

[0369] some instances, epithelial restitution is increased between 10-12%, between 11-13%, between 12-14%, or between 13-15%, compared to a control.

[0370] Epithelial wound healing involves coordinated migration and proliferation of epithelial cells. Epithelial cells adjacent to the wound migrate as a sheet to cover denuded surfaces, which is also referred to as “epithelial restitution” (Nusrat, A et al. “Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells.” The Journal of clinical investigation vol. 89,5 (1992): 1501-11, the contents of which are incorporated herein in their entirety). Such epithelial sheet migration requires dynamic and coordinated remodeling of cell-cell and cell-matrix adhesions. Epithelial cells at the leading edge extrude filamentous-actin-rich protrusions that dynamically adhere to the matrix and mediate forward movement of the epithelial sheet. Epithelial transmembrane integrins in focal adhesive contacts include β1 integrin that mediate bidirectional cross-talk between the matrix and intracellular signaling events to influence epithelial cytoskeletal restructuring and polarity required to guide epithelial movement and control cell proliferation. A number of small GTPases coordinate cellular events during wound closure. These include the Rho GTPase family member, Rac1 that controls cellular protrusions at the leading edge. Expression of constitutively active Rac1 influences epithelial movement and proliferation by targeting β1 integrin in cellular protrusions and modulating actin dynamics. In addition, formyl peptide receptor-1 (FPR1) signaling activates Rac1 and Rho kinase to regulate intestinal epithelial cell (IEC) migration and wound repair. Epithelial-derived secreted proteins that include epidermal growth factor (EGF) and transforming growth factor-β1 (TGF-β1) coordinate repair of the epithelial barrier.il However, a secreted Wnt antagonist (Dickkopf-1) Dkk-1 modifies the activation of another small GTPase, Cdc42 to influence directional orientation of migrating lECs.

[0371] The gastrointestinal epithelium produces a wide variety of peptides which may contribute to protection from injury as well as repair after injury occurs. Restitution is accomplished by rapid migration of the epithelium to re-establish surface epithelial continuity. A wide variety of growth factors and cytokines, which are produced both by the epithelium itself and by lamina propria cell populations, promote restitution in models of epithelial injury. These include members of the epidermal growth factor (EGF) / transforming growth factor (TGF)a, and the fibroblast growth factor (FGF) families, as well as a variety of cytokines (interleukin [IL]-1, IL-2, IL-4, IL- 15, and interferon y) which interact with their cognate receptors on the intestinal epithelial basolateral surface. These growth factors and cytokines appear to promote restitution through a TGFβ-dependent pathway and act to both enhance expression of TGFβ and to enhance its bioactivation. In contrast, trefoil peptides, members of 4)

[0372] a recently recognized family of small proteins produced by goblet cells, both protect the epithelium and promote restitution following secretion onto the apical surface through mechanisms distinct from those peptides acting through TGFβ. Thus, rapid repair after epithelial injury is achieved through complementary mechanisms acting at the basolateral and apical surfaces of the epithelium.

[0373] Also provided herein are methods of reducing at least one symptom of a gastrointestinal disorder or skin disorder in a subject, comprising administering an effective amount of any microbial composition or postbiotic composition disclosed herein. In some aspects, the symptom is reduced at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to a control. A control can include a subject that is not administered said microbial composition or postbiotic composition. Administration of an effective amount of a microbial composition or postbiotic composition disclosed herein may reduce inflammation in a tissue of a subject. Any tissue can exhibit reduced inflammation following administration of such compositions. In such instances, the tissue can be gastrointestinal tissue. In another instance, the tissue can be skin tissue. For instance, administration of the microbial composition or postbiotic composition may reduce inflammation at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control. Methods of measuring inflammation may be dependent upon the specific tissue, and will be readily understood in the art. For instance, expression of genes associated with inflammation and / or production of molecules associated with inflammation can be assessed. Methods for assessing gene expression and molecule levels are dependent upon the specific factor, but are readily understood in the art. Any method can be utilized. In some instances, a biopsy of tissue can be taken to visualize inflammation and / or tissue damage associated with inflammation.

[0374] Also provided herein are methods of improving well-being of a subject, comprising administering an effective amount of any microbial composition or postbiotic composition disclosed herein. In some aspects, well-being of the subject is improved at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to a control. A control can include a subject that is not administered said microbial composition or postbiotic composition.

[0375] In some aspects, said method improves the quality of life of a subject as measured using the SF-36 questionnaire. In some aspects, said SF-36 questionnaire score of said subject increases at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, or at least 2.0-fold compared to a control. 4)

[0376] In some aspects, said method reduces anxiety in a subject as measured by the

[0377] generalized anxiety disorder assessment (GAD-7). In some aspects, said GAD-7 score of said subject is reduced at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to a control.

[0378] Also provided herein are methods of increasing levels of protocatechuic acid (PCA) in a subject. In some instances, PCA levels are increased in a tissue of a subject. Such methods comprises administering an effective amount of any microbial composition or postbiotic composition disclosed herein. Such compositions would be expected to be capable of

[0379] delivering PCA to a target tissue. Any tissue can be targeted for administration of the microbial and postbiotic compositions disclosed herein. In some instances, the tissue can be

[0380] gastrointestinal tissue. In another instance, the tissue can be skin tissue. In particular, the microbial compositions disclosed herein may deliver a viable engineered S. boulardii strain described herein to a tissue. In some instances, the engineered S. boulardii strain is capable of producing PCA. Without wishing to be bound by any theory, administration of such engineered S. boulardii may result in establishment of a plurality in the gastrointestinal tract of a subject.

[0381] The presence of a plurality of engineered S. boulardii may produce and secrete PCA into the gastrointestinal tract of a subject. The postbiotic composition disclosed herein may comprise PCA, as described elsewhere herein. Such postbiotic composition may deliver such levels of

[0382] PCA to a target tissue. In some aspects, levels of PCA in a tissue of the subject is increased at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to a control. A control can include a subject that is not administered said microbial composition or postbiotic composition.

[0383] As used herein, an “increase in” or “increasing” PCA production comprises any statistically significant increase the level of PCA when compared to an appropriate control.

[0384] Such increases can include, for example, at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or greater increase in the PCA level. Such increases can also include, for example, at least about a 3%-l 5%, 10%-25%, 20% to 35%, 30% to 45%, 40%-55%, 50%-65%, 60%-75%, 70%-85%, 80%-95%, 90%-105%, 100%- 115%, 105%- 120%,

[0385] 115% -130%, 125%-150%, 140%-160%, 155%-500% or greater increase in the PCA level. The level of PCA can be measured in a sample taken from a subject, or measured directly in the subject. In specific embodiments, the level of PCA is measured in an intestine, colon, skin, blood, urine, or fecal sample taken from a subject, such as a human or milk-producing 4)

[0386] agricultural animal. In specific embodiments an increase in PCA production is measured in reference to the level of PCA in a control sample. As used herein a control includes but is not limited to, the level of PCA in a corresponding sample from a subject that was not administered the engineered S. boulardii strain and / or cell constituent(s) thereof, the level of PCA in a sample from the subject prior to administration of the S. boulardii strain and / or cell constituents thereof, or the level of PCA in a standardized sample from a subject that was not administered S. boulardii strain and / or cell constituents thereof. One of skill in the art would be able to identify proper controls in order to measure an increase in the level of PCA. PCA can be measured by high performance liquid chromatography (HPLC). One can detect PCA using a reverse-phase gradient of mobile phase consisting of an aqueous phase such as water with or without modifiers and an organic solvent such as methanol or acetonitrile. The mobile phase is pumped across a solid phase such as a Cl 8 column. Compounds dissociating from the solid phase are detected by DAD (Diode Array Detection) or other means, at various UV wavelengths and retention times. PCA can be positively identified by comparison of the retention time and UV spectrum to authentic chemical standards. See, for example, Assefa et al. (BMC Chem 13:56 (2019), herein incorporated by reference. In specific embodiments, the level of PCA from a sample collected from a subject or collected in vitro following administration of S. boulardii strain and / or cell constituents thereof will not demonstrate an increase in PCA production.

[0387] Methods of Administration

[0388] Methods are provided herein for altering or maintaining at least one biological activity in a subject with an effective amount of a composition comprising Saccharomyces cerevisiae var. boulardii and / or cell constituents thereof. The methods comprise administering an effective amount of a composition comprising Saccharomyces cerevisiae var. boulardii and / or cell constituents thereof. These methods can be performed in vitro, in vivo, or ex vivo. When performed in vivo, the method comprises administering to a subject an effective amount of any composition disclosed herein. The compositions comprise Saccharomyces cerevisiae var. boulardii and / or cell constituents thereof, wherein said S. boulardii comprises a heterologous polynucleotide encoding a 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or comprises a conservatively substituted version of SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. 4)

[0389] In some embodiments, an effective amount of the composition alters or maintains at least one biological activity in a subject. In such instances, an effective amount of the composition can: i) suppress expression of at least one gene associated with inflammation in at least one cell present in said subject; ii) stimulate an epithelial tissue response in said subject; iii) reduce at least one symptom of a gastrointestinal or skin disorder in said subject; and / or iv) improve well-being of said subject, compared to a control, wherein said control comprises a subject who is not administered an effective amount of said composition.

[0390] An effective amount of the composition is determined based on the intended goal. The term "unit dose" refers to a physically discrete unit suitable for use in a subject or agricultural animal, each unit containing a predetermined quantity of the yeast and / or cell constituents thereof calculated to produce the desired response in association with its administration, i.e., the appropriate route and dosage regimen. The quantity to be administered, both according to number of administrations and unit dose, depends on the subject to be treated, the state of the subject, the environmental conditions of the subject, and the result desired. Precise amounts of the yeast strains and / or cell constituents thereof also depend on the judgment of the practitioner and can be unique to each individual.

[0391] In specific embodiments, the effective amount of a Saccharomyces cerevisiae var. boulardii disclosed herein is about 104CFU to about 1012CFU, about 105CFU to about 1012CFU, about 106CFU to about 1012CFU, about 107CFU to about 1012CFU, about 108CFU to about 1012CFU, about 109CFU to about 1012CFU, about IO10CFU to about 1012CFU, about 1011CFU to about 1012CFU, about 105CFU to about 1011CFU, about 105CFU to about 1011CFU, about 106CFU to about 1011CFU, about 107CFU to about 1011CFU, about 108CFU to about 1011CFU, about 109CFU to about 1011CFU, about IO10CFU to about 1011CFU, about 105CFU to about IO10CFU, about 106CFU to about IO10CFU, about 107CFU to about IO10CFU, about 108CFU to about IO10CFU, or about 109CFU to about IO10CFU. In other embodiments, the effective amount is at least about 104CFU, at least about 105CFU, at least about 106CFU, at least about 107CFU, at least about 108CFU, at least about 109CFU, at least about IO10CFU, at least about 1011CFU, or at least about 1012CFU.

[0392] In some embodiments, a Saccharomyces cerevisiae var. boulardii and / or cell constituents thereof is administered to a subject that is also administered (simultaneously or sequentially) a prebiotic. Prebiotics, such as those described herein, may be combined with a strain disclosed herein into a formulated product or the prebiotic(s) may be administered separately from (before, during, or after) a bacterial strain disclosed herein. 4)

[0393] In some embodiments, the supernatant of a Saccharomyces cerevisiae var. boulardii culture disclosed herein is administered to a subject. Such supernatants are cell-free. The term “cell free” when used to refer to a supernatant means that the supernatant comprises no more than: i) 1000 CFU / gram, 100 CFU / gram, 10 CFU / gram, or 1 CFU / gram; and / or h) 1000 CFU / ml, 100 CFU / ml, 10 CFU / ml, or 1 CFU / ml, of said Saccharomyces cerevisiae var. boulardii. Methods for generating cell-free culture supernatants are readily known in the art. For instance, a cell culture can be centrifuged to separate yeast cells. A cell culture can also be subjected to filter-sterilization to remove yeast cells. Any method known in the art is envisaged within the scope of the present disclosure for generating a cell-free culture supernatant.

[0394] In some embodiments, the supernatant is administered simultaneously or sequentially with one or more compositions disclosed herein. The supernatant may be combined with a strain disclosed herein into a formulated product or the supernatant may be administered separately from (before, during, or after) a Saccharomyces cerevisiae var. boulardii strain disclosed herein.

[0395] In those embodiments wherein the prebiotic and / or supernatant is administered separately from composition comprising Saccharomyces cerevisiae var. boulardii, the composition may be administered before, during, or after the prebiotic and / or supernatant. The yeast strain and the additional component can be administered to a subject within minutes (e.g., 1, 2, 5, 10, 15, 30, 45 minutes), hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 hours), or days (e.g., 1, 2, 3, 4, 5, 6, 7 days) of each other.

[0396] As used herein, the compositions can be used to alter and / or maintain at least one biological activity in any subject when compared to an appropriate control (e.g., a sample from the subject prior to administration of the composition). By “subject’ is intended animals. In specific embodiments, the subjects are mammals, e.g., primates or humans.

[0397] The compositions disclosed herein can be administered to increase PCA levels in a subject. PC A levels can be increased in any tissue. For instance, following oral administration, PCA levels may be increased in the gastrointestinal tract. Alternatively, following topical administration, PCA levels may be increased on the skin.

[0398] As used herein, an “increase in”, or “increasing” PCA levels comprises any statistically significant increase in the level of PCA when compared to an appropriate control (e.g., a corresponding sample not contacted with a composition). Such increases can include, for example, at least a 5%, 10%, 20%, 30%, 40%, 50% or more increase in PCA levels when compared to an appropriate control. Such increases can also include, for example, at least about a 1-15%, 10-25%, 20-35%, 30-45%, 40- 4)

[0399] 55% or more increase in PC A levels when compared to an appropriate control. PCA levels can be measured in a Saccharomyces cerevisiae var. boulardii composition. In some embodiments, PCA levels can be measured in a sample taken from a subject or measured directly in the subject.

[0400] In specific embodiments, an increase in PCA levels is measured in reference to the level of PCA in a control sample. As used herein, a control includes but is not limited to, the level of PCA in a corresponding sample that does not comprise a yeast strain disclosed herein, the level of PCA from a subject that was not administered a composition disclosed herein, the level of PCA in a sample from the subject prior to administration of a composition disclosed herein, or the level of PCA in a standardized sample from a subject that was not administered a composition disclosed herein. One of skill in the art would be able to identify proper controls in order to measure an increase in the level of PCA in a tissue of a subject.

[0401] Thus, the presently disclosed methods comprise increasing PCA levels in a tissue of a subject when compared to an appropriate control by administering an effective amount of a composition comprising a Saccharomyces cerevisiae var. boulardii strain disclosed herein and / or cell constituents thereof. An appropriate control could be, but is not limited to the level of PCA in a corresponding sample that does not comprise the Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof, the level of PCA in a corresponding sample from a subject that was not administered the Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof, the level of PCA in a sample from the subject prior to administration of the Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof, and / or the level of PCA in a standardized sample from a subject that was not administered a composition comprising the Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof.

[0402] In some embodiments, the composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof increases the amount of PCA in a subject by at least 10%. In some embodiments, the composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof increases the amount of PCA in a subject by at least 20%. In some embodiments, the composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof increases the amount of PCA in a subject by at least 30%. In some embodiments, the composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof increases the amount of PCA in a subject by at least 40%. In some embodiments, the composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof increases the amount of PCA in a subject by at least 50%. In some embodiments, the 4)

[0403] composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof increases the amount of PCA in a subject by at least 60%. In some embodiments, the composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof increases the amount of PCA in a subject by at least 70%. In some embodiments, the composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof increases the amount of PCA in a subject by at least 80%. In some embodiments, the composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof increases the amount of PCA in a subject by at least 90%. In some embodiments, the composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof increases the amount of PCA in a subject by more than 90%.

[0404] PCA can be readily identified by methods known in the art, including high-performance liquid chromatography (HPLC) and / or mass spectrometry.

[0405] The composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof can be administered to a subject based on standard techniques known in the art for administration to the particular type of subject and in the environment in which the subject receives the composition. When administered to a human, the composition may be a liquid formulation or a solid formulation. The solid formulation can have PCA present. The PCA can be present in the solid formulation at any concentration provided in Tables 9-11. Additionally, the liquid formulation can have PCA present. The PCA can be present in the liquid formulation at any concentration provided in Tables 9-11. The composition can be administered mucosally via oral administration, nasal administration, or rectal administration, for example, or administered parenterally, including but not limited to subcutaneous administration, transdermal administration, or any method that allows the composition to maintain and / or alter at least one biological activity in a subject.

[0406] It would be understood that administration can be dependent upon an amount or concentration of PCA.

[0407] In certain embodiments, the strain with these characteristics is a deposited strain. In some instances, the strain is deposited under Accession No. X.

[0408] In some embodiments of the invention, the method comprises administration of multiple doses of a composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof to a subject. The method may comprise administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more effective doses of such a composition. In some embodiments, doses are administered over the course of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 4)

[0409] 21 days, 30 days, or more than 30 days. The frequency and duration of administration of multiple doses of the compositions is such as to maintain and / or alter at least one biological activity in a subject when compared to an appropriate control. It will also be appreciated that the effective amount or dosage of a composition comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays for detecting PCA levels and / or at least one biological activity in a subject known in the art and described herein. As used herein, “treatment” or “treating” refers to therapeutic (e.g., curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the condition or the symptoms of a subject) and preventative effects. The prophylactic administration (wherein a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof is administered in advance of symptoms) serves to prevent or attenuate any subsequent symptom. When provided therapeutically, the disclosed compositions are provided at (or shortly after) the onset of a symptom. The therapeutic administration of the substance may serve to attenuate any actual symptom.

[0410] In one aspect, compositions and formulations disclosed herein are applied topically (i.e., as a topical application) to a subject. In some embodiments, the composition applied topically comprises a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof. In some embodiments, the composition applied topically comprises a supernatant of a Saccharomyces cerevisiae var. boulardii culture. In some embodiments, the composition applied topically comprises comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof in combination with a prebiotic. The compositions and formulations applied topically may further comprise a postbiotic.

[0411] In another aspect, compositions and formulations disclosed herein are administered orally (e.g., the compositions can be formulated into any food and / or beverage product described herein) to a subject. In some embodiments, the composition administered orally comprises a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof. In some embodiments, the composition administered orally comprises a supernatant of a Saccharomyces cerevisiae var. boulardii culture. In some embodiments, the composition administered orally comprises comprising a Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof in combination with a prebiotic. The compositions and formulations administered orally may further comprise a postbiotic.

[0412] In some aspects, the disclosure provides methods of using host cells for producing PCA, one or more precursors of PCA, or a product for which PCA is a precursor. In some instances, PCA is converted into a second molecule. Said second molecule can be a molecule for which PCA is a 4)

[0413] precursor. In some instances, said second molecule is a naturally occurring molecule, for which PCA is a precursor. In some instances, said second molecule is a synthetic molecule. In some embodiments, the disclosure provides a method comprising the steps of: culturing a host cell described in this application (e.g., a host cell comprising a heterologous polynucleotide encoding an enzyme associated with the present disclosure). Methods for culturing cells are described elsewhere in this application. In some embodiments, the disclosure provides a method of producing PCA, one or more precursors of PCA, or a product for which PCA is a precursor comprising culturing a host cell described in this application (e.g., a host cell comprising a heterologous polynucleotide encoding an enzyme associated with the present disclosure). In some embodiments, the production occurs ex vivo, e.g., in an in vitro cell culture environment. Compositions, cells, enzymes, and methods described in this application are also applicable to industrial settings, including any application wherein there is a need for increased biosynthesis of PCA, one or more precursors of PCA, or a product for which PCA.

[0414] In some embodiments, methods associated with the disclosure include methods of producing one or more of the following products: PCA, vanillin, muconic acid, adipic acid, precursors or derivatives thereof.

[0415] Any molecule for which PCA is a precursor is envisaged as within the scope of being produced using the methods of producing PCA described herein.

[0416] In particular, the PCA produced by the engineered S. boulardii strain can be a chemical intermediate. PCA is versatile as a building block in synthesizing a wide-range of high-value chemicals and bioactive compounds. PCA's unique structure, containing both hydroxyl and carboxyl functional groups, makes it an ideal precursor for producing pharmaceutical actives, agrochemicals, flavors, fragrances, and specialty polymers. This versatility allows industries to leverage PCA not only in traditional drug development but also in green chemistry applications, where sustainable and bio-based intermediates are increasingly replacing petroleum-derived inputs. Furthermore, with the rising global demand for natural and semi-synthetic molecules, manufacturers are investing heavily in PCA-based intermediates to reduce costs and increase process efficiency. In some instances, the microbial cultures and PCA produced therefrom of the present disclosure can be integrated into a method for producing any chemical molecule, including, but not limited to, pharmaceutical actives, agrochemicals, flavors, fragrances, and specialty polymers, for which PCA serves as a suitable and effective precursor molecule. Molecules for which PCA would serve as effective precursor would be readily understood in the art. As such, the production and use of PCA as a chemical intermediate for producing downstream molecules is well-established in the art. In some aspects, the disclosure provides methods of 4)

[0417] engineering a host cell to express one or more heterologous enzymes capable of increasing production of PC A, one or more precursors of PCA, or a product for which PCA is a precursor. In some embodiments, a biochemical pathway for the production of PCA is provided in Figure 4. In some embodiments, the expression of various enzymes in a biochemical pathway for the production of PCA are variously increased (or introduced) or decreased, altered or substituted. In some embodiments, an enzyme of the one or more heterologous enzymes is AroZ, Arol, Aro3, Aro4, Tkll, Tall, AroB, AroD, or AroF. In some embodiments, one or more of the one or more enzymes is a member of shikimate pathway. In some embodiments, Arol, Aro3, and Aro4 produce increased amounts of one or more precursors of PCA. In some embodiments, Arol, Aro3, and Aro4 convert one or more products of the pentose phosphate pathway to 3-dehydroshikimate. In some embodiments, Arol, Aro3, and Aro4 produce increased amounts of 3-dehydroshikimate. In some embodiments, AroZ converts 3-dehydroshikimate to protocatechuic acid (PCA). In some embodiments, in a host cell producing PCA, various additional genetic modifications can be made to increase PCA production. In some embodiments, these additional genetic modifications include the deletion of (or decreased expression, activity or levels of) genes which relate to unproductive branches of the biochemical pathway, such as zwfl or aroE; or genes that convert PCA to unwanted products, such as PobA, AroY and GDC1.

[0418] In some instances, the engineered S. boulardii strains disclosed herein are further engineered to enhance production and / or tolerance of PCA. Biochemical synthesis pathways, and associated enzymes, that would enhance PCA production in such strains are readily known in the art. For instance, FIG.4 provides a diagram depicting a biochemical pathway which may enable an engineered microorganism to increase production of PCA. In some instances, S. boulardii is engineered to inhibit a signaling pathway that reduces the efficacy of the PCA production pathway. For instance, a microorganism, such as a yeast strain, including an engineered S. boulardii disclosed herein, which comprise a heterologous polynucleotide encoding a 3-dehydroshikimate dehydratase (AroZ) or an active variant thereof, may be further engineered to enhance PCA production via utilization of glucose. Such strains may comprise additional features that enhance PCA production and / or tolerance. In some instances, the S. boulardii strain may lack at least one enzyme, wherein the absence of said enzyme enhances production or tolerance of PCA. For example, such strains may lack a functional AroE enzyme, or polynucleotide sequence encoding such an enzyme,

[0419] The engineered S. boulardii strains disclosed herein which are capable of producing high purity PCA, can be used to produce any downstream molecule known in the art. In such instances, PCA can be produced as an intermediate molecule through fermentation of an engineered S. boulardii disclosed 4)

[0420] herein. In some instances, the high-purity PCA produced from the disclosed microbial cultures is purified and utilized to produce a second, downstream molecule.

[0421] In some embodiments, an enzyme associated with the present disclosure may comprise one or more modifications to enhance its effectiveness (e.g., activity and / or stability (e.g., half-life)) in a selected mode of biosynthesis. For example, an enzyme associated with the present disclosure may comprise a modification that increases stability and / or activity of the enzyme at acidic pH, e.g., to improve the effectiveness of the enzyme when used in an industry-level batch culture. In some embodiments, the enzyme is immobilized to another agent, e.g., a different enzyme, a polymer (e.g., polysaccharide (e.g., starch)), or an inorganic carrier (e.g., silica gel). Immobilization may increase enzyme stability and / or shelf-life.

[0422] Compositions

[0423] Further aspects of the disclosure relate to compositions containing PCA, one or more precursors of PCA, or a product for which PCA is a precursor. Culturing of host cells associated with the disclosure can result in compositions comprising products, including PCA, one or more precursors of PCA, or a product for which PCA is a precursor. In some embodiments, compositions obtained by culturing host cells associated with the disclosure result in compositions in which at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the total products in the composition is / are PCA, one or more precursors of PCA, or a product for which PCA is a precursor.

[0424] PCA can be present in any composition disclosed herein at any concentration depicted in Tables 9-11.

[0425] Compositions associated with the disclosure can further comprise additional components as would be understood by one of ordinary skill in the art. For example, it should be appreciated that in some embodiments, compositions comprising PCA, one or more precursors of PCA, or a product for which PCA is a precursor can include cell culture fermentation broth or cell culture supernatants. In other embodiments, compositions may include PCA, one or more precursors of PCA, or a product for which PCA is a precursor in a form that has been purified from cell culture fermentation broth or cell culture supernatants. 4)

[0426] Further aspects of the disclosure relate to compositions comprising host cells described herein. In some embodiments, a composition comprises a host cell that expresses one or more heterologous polynucleotides that encode one or more enzymes associated with the present disclosure. In some embodiments, a composition is a probiotic. In some embodiments, the composition comprises one or more probiotic strains. Non-limiting examples of probiotic strains include the genera, species and strains: Bacillus, Bacillus coagulans, Bacillus subtilis, Bifidobacterium, Bifidobacterium adolescentis, Bifidobacterium adolescents SD-BA5-IT, Bifidobacterium animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. lactis DN-173 010, Bifidus regularis, Bifidobacterium bifidum, Bifidobacterium bifidum Bb-06, Bifidobacterium bifidum UABb-10, Bifidobacterium breve. Bifidobacterium breve HRVD521-US, Bifidobacterium breve SD-BR3-IT, Bifidobacterium breve Bb-18, Bifidobacterium infantis, Bifidobacterium infantis SD-M63-JP, Bifidobacterium lactis, Bifidobacterium lactis DN-173 010, Bifidobacterium lactis HRVD524-US, Bifidobacterium lactis SD-BS5-IT, Bifidobacterium lactis SD-CECT8145-SP, Bifidobacterium lactis SD-MB2409-IT, Bifidobacterium lactis SD150-BE, Bifidobacterium lactis Bl-04, Bifidobacterium lactis, HN019, Bifidobacterium longum, Bifidobacterium longum HRVD90b-US, Bifidobacterium longum SD-BB536-JP, Bifidobacterium longum SD-CECT7347-SP, Bifidobacterium longum BI-05, Bifidobacterium longum subsp. longum 35624, Bifantis, Enterococcus, Escherichia, Escherichia colt, Escherichia colt Nissle 1917, Lacticaseibacillus, Lacticaseibacillus casei, Lacticaseibacillus casei HRVD300-US, Lacticaseibacillus casei SD-CECT9104-SP, Lactobacillus casei Shirota, Lactobacillus casei Lc-11, Lacticaseibacillus rhamnosus, Lacticaseibacillus rhamnosus HRVD113-US, Lacticaseibacillus rhamnosus SD-GG-BE, Lacticaseibacillus rhamnosus SD-LR6-IT, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lr-32, Lactiplantibacillus plantarum, Lactiplantibacillus plantarum SD-LP1-IT, Lactiplantibacillus plantarum SD-LPLDL-UK, Lactiplantibacillus plantarum Lp-115, Lactobacillus, Lactobacillus acidophilus, Lactobacillus acidophilus UALa-01, Lactobacillus acidophilus La- 14, Lactobacillus brevis, Lactobacillus brevis Lbr-35, Lactobacillus casei rhamnosus. Lactobacillus casei rhamnosus Lcr35, Lactobacillus crispatus, Lactobacillus crispatus SD-LCR01-IT, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Limosilactobacillus fermentum, Limosilactobacillus fermentum SBS-1, Lactobacillus gasseri, Lactobacillus gasseri Lg-36, Lacticaseibacillus paracasei, Lacticaseibacillus paracasei Lpc-37, Limosilactobacillus reuteri, Limosilactobacillus reuteri 1E1, Ligilactobacillus, Ligilactobacillus salivarius, Ligilactobacillus salivarius SD-LS1-IT, Ligilactobacillus salivarius Ls-33, Limosilactobaciilus, Limosilactobacillus fermentum, Limosilactobacillus fermentum SD-LF8-IT, Limosilactobacillus reuteri. 4)

[0427] Limosilactobacillus reuteri RD830-FR, Limosilactobacillus reuteri SD-LRE2-IT, Pediococcus, Saccharomyces, Saccharomyces cerevisiae var. boulardii, Streptococcus, and Streptococcus thermophilus.

[0428] The term “probiotic” refers to a microbial cell preparation or components of microbial cells with beneficial effects on the health and / or well-being of a subject. In some embodiments, a probiotic is one or more live microorganisms that, when administered in a sufficient amount to a subject, confer a health benefit to the subject.

[0429] In some embodiments, a probiotic host cell refers to any consumable host cell that provides health benefits to a subject when consumed. Probiotics are considered to be generally safe and help restore the balance of intestinal flora, keep it stable by positively changing the composition of the intestinal flora of humans and animals, and / or positively affect the part of the immune system which communicates with the intestinal wall. Through the production of metabolites, such as acetic acid, lactic acid and hydrogen peroxide, probiotic microorganisms, for example, deteriorate the living conditions of undesirable microorganisms in the gut. The presence of probiotic microorganisms in the gut improves the digestion function and can both be used in a therapeutic set-up for example to treat gastrointestinal disorders as diarrhea or in a preventive set-up for example to maintain a well- balanced gut microbiome and gastrointestinal comfort. A "probiotic additive" or equivalently "probiotic supplement" refers to a substance in any shape or form that contains probiotics. More specifically, a probiotic substance can be dry or liquid and comprises live probiotics embedded in a matrix of sugars, proteins and / or polysaccharides.

[0430] In some embodiments, a probiotic composition is a probiotic dietary supplement. The term “probiotic dietary supplement” refers to a dietary supplement comprising a probiotic host cell. In some embodiments, a probiotic dietary supplement further comprises one or more of one or more amino acids, caffeine, cappuccino, a carbohydrate, a cereal product, a flavoring (e.g., chocolate), coffee, a coloring agent, a culinary product, fiber or dietary fiber, a lipid, a fat, malt, ice cream, milk, a milk product, a nutritional powder, a nutritional liquid, a protein, a ready-drink formulation, syrup, water, and / or yogurt. In some embodiments, a probiotic dietary supplement further comprises one or more of adsorbents, antimicrobials, antioxidants, binders, carriers, co-compounds, coatings, dispersing agents, emulsifiers, encapsulating agents / materials, fillers, film forming agents, flowing agents, gel forming agents, hydrocolloids (such as gums, proteins, and / or modified starches), jellifying agents, matrix compounds, processing aids (solvents), solubilizing agents (oils, fats, waxes, and / or lecithins, etc.), surface active agents, taste masking agents, wall / shell materials, weighting agents, and / or wetting 4)

[0431] agents. In some embodiments, a probiotic dietary supplement further comprises one or more of an additive, an adjuvant, a buffer, a colorant, a diluent, an emulsifying agent, an excipient, a filler, a flavoring agent, a gelatin, gum arabic, lignin sulfonate, a lubricant, magnesium (e.g., magnesium citrate); a metal ion; a mineral; polyalkylene glycol, a preservative, a stabilizer, a starch, a sugar, a talc, a vegetable gum, a vegetable oil, water, a wetting agent, and / or xylitol.

[0432] In some embodiments, a composition further comprises a pharmaceutically acceptable excipient. In some embodiments, a composition further comprises a vehicle that is generally recognized as safe.

[0433] Formulations of S. boulardii

[0434] The S. boulardii microbial compositions and S. boulardii-derived postbiotic compositions disclosed herein are particularly effective at maintaining and / or altering at least one biological activity in a subject. Said compositions can be particularly effective when consumed by an individual. In such instances, any formulation of the microbial and postbiotic compositions disclosed herein that promote consumption by an individual will be desirable. For instance, it may be advantageous to include the microbial and / or postbiotic compositions within a food or beverage product for more efficient and effective consumption by an individual and / or delivery to the gastrointestinal tract. In an alternative instance, said compositions can be particularly effective when topically applied to an individual. In such instances, any formulation of the microbial and postbiotic compositions disclosed herein that enables topical administration to an individual is desirable. In such aspects, it can be advantageous to include the microbial and / or postbiotic compositions within a paste (e.g., a cell paste), a powder, dust, a slurry, aqueous or oil-based liquid products, and / or gel.

[0435] Disclosed herein are food and beverage products comprising the microbial compositions and postbiotic compositions of the present disclosure. The compositions can be incorporated into a food or beverage produce in any form, such as, but not limited to, as a gel, paste, tablet, powder, gummy, spray, aerosol, lozenge, effervescent tablet, orodispersible film, topical cream, ointment, suppository, emulsion, transdermal patch, or liquid. In a particular instance, the composition can be incorporated into a powder. In such instances, a powder can be suspended into a liquid food or beverage product.

[0436] The food or beverage product can be any food or beverage product intended for human or animal consumption, non-limiting examples of which include a nutritional whole food, drink, sports drink, coffee, tea, water, mineral water, soda, carbonated beverage, oral hydration drink, mineral water, soup, replacement food, nutritional bar, nutritional consumable, gummy, tablet, confectionery, 4)

[0437] fermented or unfermented milk-based product, yogurt product, milk-based powder, enteral nutritional product, fermented or unfermented cereal-based product, chocolate, milk, yogurt, cheese, ice cream, baby and infant formula, cereal, animal feed, protein shake, protein powder, functional water juice blend, smoothie, ready-to-drink (RTD) beverage, non-dairy milk, nut or seed butter, energy drink, functional shot, baked good, granola bar, snack bar, alcoholic beverage, frozen food, frozen meal, condiment, pasta, noodles, pet food, or pet treat.

[0438] In some embodiments, the food or beverage product comprises juice, water, milk, a liquid containing protein, or tea. In some embodiments, the food or beverage product comprises a semisolid, such as yogurt, or a solid, such as gelatin. In some embodiments, the food or beverage product comprises a powder intended to be mixed with a fluid.

[0439] The food or beverage products can be formulated with various amounts of the microbial composition or postbiotic composition. In some instances, the food or beverage product is a liquid, such as a juice, a sports drink, a smoothie, a protein shake, water or tea. The food or beverage product can also be a solid, or semi-solid. For example, the food or beverage product can be a gummy or a tablet. In some embodiments, the tablet is a solid tablet. In some embodiments, the tablet is a chewable tablet. In some embodiments, the microbial composition or postbiotic composition comprises a powder, such as a lyophilized powder. When the microbial composition or postbiotic composition comprises a powder, the powder can be added to a liquid food or beverage product.

[0440] The food or beverage produce can comprise an engineered S. boulardii strain disclosed herein at a concentration of at least about 104CFU / g to about 1012CFU / g, about 105CFU / g to about 1012CFU / g, about 106CFU / g to about 1012CFU / g, about 107CFU / g to about 1012CFU / g, about 108CFU / g to about 1012CFU / g, about 109CFU / g to about 1012CFU / g, about IO10CFU / g to about 1012CFU / g, about 1011CFU / g to about 1012CFU / g, about 105CFU / g to about 1011CFU / g, about 105CFU / g to about 1011CFU / g, about 106CFU / g to about 1011CFU / g, about 107CFU / g to about 1011CFU / g, about 108CFU / g to about 1011CFU / g, about 109CFU / g to about 1011CFU / g, about IO10CFU / g to about 1011CFU / g, about 105CFU / g to about IO10CFU / g, about 106CFU / g to about IO10CFU / g, about 107CFU / g to about IO10CFU / g, about 108CFU / g to about IO10CFU / g, or about 109CFU / g to about IO10CFU / g. In other embodiments, the concentration of Saccharomyces cerevisiae var. boulardii incorporated into a food or beverage product disclosed herein comprises or consists of at least about 104CFU / g, at least about 105CFU / g, at least about 106CFU / g, at least about 107CFU / g, at least about 108CFU / g, at least about 109CFU / g, at least about IO10CFU / g, at least about 1011CFU / g, or at least about 1012CFU / g. Another such food or beverage product can comprise a concentration of 4)

[0441] engineered S. boulardii of at least about 104CFU / ml to about 1012CFU / ml, about 105CFU / ml to about 1012CFU / ml, about 106CFU / ml to about 1012CFU / ml, about 107CFU / ml to about 1012CFU / ml, about 108CFU / ml to about 1012CFU / ml, about 109CFU / ml to about 1012CFU / ml, about IO10CFU / ml to about 1012CFU / ml, about 1011CFU / ml to about 1012CFU / ml, about 105CFU / ml to about

[0442] 1011CFU / ml, about 105CFU / ml to about 1011CFU / ml, about 106CFU / ml to about 1011CFU / ml, about 107CFU / ml to about 1011CFU / ml, about 108CFU / ml to about 1011CFU / ml, about 109CFU / ml to about 1011CFU / ml, about IO10CFU / ml to about 1011CFU / ml, about 105CFU / ml to about

[0443] IO10CFU / ml, about 106CFU / ml to about IO10CFU / ml, about 107CFU / ml to about IO10CFU / ml, about 108CFU / ml to about IO10CFU / ml, or about 109CFU / ml to about IO10CFU / ml. In other embodiments, the concentration of S. boulardii comprises or consists of at least about 104CFU / ml, at least about 105CFU / ml, at least about 106CFU / ml, at least about 107CFU / ml, at least about 108CFU / ml, at least about 109CFU / ml, at least about IO10CFU / ml, at least about 1011CFU / ml, or at least about 1012CFU / ml.

[0444] Such food and beverage products can further comprise PCA in any concentration disclosed herein. In some instances, PCA is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% weight per volume (w / v), or volume per volume (v / v), of the food or beverage product. In some instances, PCA is present in the food or beverage product at a concentration of at least 0.0005 mM (i.e., 0.5 uM), 0.001 mM (i.e., 1 uM), at least 0.005 mM. at least 0.01 mM, at least 0.03 mM, at least 0.06 mM, at least 0.12 mM, at least 0.25 mM, at least 0.5 m, at least I M, at least 2 mM, at least 5 mM, or more than 5 m In certain instances, PCA is present at a concentration of between about 0.5(uM to about 5 gM (i.e, 0.005 mM), about I. M4o about 10 u (i.e.. 0.01 mM), about 0.005 nM to about 0.03 mM, about 0.01 mM:;. about 0.06 mM, about 0.03 mM to about 0.12 mM, or about 0.06 M to about 0.25 mM.

[0445] PCA can be present in the food or beverage product at a concentration of between about 0.001 mM to about 0.01 mM, about 0.002 mM to about 0.01 mM, about 0.003 mM to about 0.01 mM. about 0.004 mM to about 0.01 mM, about 0.005 rnM to about 0.01 mM, about 0.006 mM to about 0.01 mM, about 0.007 mM to about 0.01 M, about 0.008 mMto about 0.01 mM, or about 0.009 mMto about 0.01 mM. In other instances, PCA is present at a concentration of between about 0.01 m to about 0.03 mM, about 0.011 mM to about 0.03 mM, about 0.012 mM to about 0.03 mM, about 0.013 mM to about 0.03 mM, about 0.014 mM to about 0.03 mM, about 0.015 mM to about 0.03 mM, about 0.016 4)

[0446] mM to about 0.03 mM, about 0.017 mMto about 0.03 mM, about 0.018 M to about 0.03 mM, about 0.019 mM to about 0.03 mM, about 0.02 mM to about 0.03 mM, about 0.022 M to about 0.03 M, about 0.023 mMto about 0.03 mM, about 0.024 m to about 0.03 m, about 0.025 mM to about 0.03 mM, about 0.026 mM to about 0.03 m, about 0.027 mMto about 0.03 mM, about 0.028 to about 0.03 M, about 0.029 mM to about 0.03 mM.

[0447] In some instances, PCA is present at a concentration of about 0.12 mM. In some instances, PCA is present at a concentration of about 0.03 mM. In some instances, PCA is present at a concentration of about 0.01 mM. In some instances, PCA is present at a concentration of about 0.005 m.

[0448] It would be understood that the concentration of PCA formulated into a food or beverage product can depend upon the dose of PCA to be administered. For instance, it may be beneficial to administer between 10 mg to 30 mg, between 20 mg to 40 mg, between 30 mg to 50 mg, between 40 mg to 60 mg, between 50 mg to 70 mg, between 60 mg to 80 mg, between 70 nig to 90 mg, between 80 mg to 100 mg, between 90 mg to 110 mg, between 100 nig to 120 nig, between 110 mg to 130 mg, between 120 mg to 140 mg, between 130 mg to 150 nig, between 140 mg to 160 mg, between 150 mg to 170 mg, between 160 mg to 180 mg, between 170 nig to 190 mg, between 180 mg to 200 mg, between 190 mg to 210 mg, between 200 mg to 220 mg, between 210 mg to 230 mg, between 220 mg to 240 mg, between 230 mg to 250 mg, between 240 mg to 260 mg, between 250 mg to 270 mg, between 260 mg to 280 mg, between 270 mg to 290 mg, between 280 mg to 300 mg, between 290 mg to 310 mg, between 300 mg to 320 mg, between 310 mg to 330 mg, between 320 mg to 340 mg, between 330 mg to 350 mg, between 340 mg to 360 mg, between 350 mg to 370 mg, between 360 mg to 380 mg, between 370 mg to 390 mg, between 380 mg to 400 mg, between 390 mg to 410 mg, between 400 mg to 420 mg, between 410 mg to 430 mg, between 420 mg to 440 mg. between 430 mg to 450 mg, between 440 mg to 460 mg, between 450 mg to 470 mg, between 460 mg to 480 mg, between 470 mg to 490 mg, between 480 mg to 500 mg of PCA per administration, or more than 500 mg of PCA per administration. It may be desired to administer more than 500 mg of PCA per administration (e.g., per day, or orally per day) It would be understood that a desired amount of PCA can be formulated imo any food or beverage product to adhere to a suitable amount of administration. For instance, the above amounts of PCA may be formulated into a single serving of any food or beverage product. The above amounts may be formulated into multiple servings of any food or beverage product. As such any food or beverage product disclosed herein can be formulated to deliver a desired amount of PCA (e.g., a desired amount of PCA in a single serving). Any formulation 4)

[0449] disclosed herein (e.g., for oral or topical administration) can be formulated in order to administer an effective amount of PCA.

[0450] PCA can be present in any food or beverage product disclosed herein at a concentration depicted in any one of Tables 9-11.

[0451] The use of PCA compositions in food packaging as an effective food preservative has been demonstrated (Zhong et ah. Peng-Fei Hou. Ya-Xing Li. Wen-Yu Yang, Mei Shu, Guo-Ping Wu, Characterization, antioxidant and antibacterial activities of gelatin film incorporated with protocatechuic acid and its application on beef preservation, LWT, Volume 151, 2021, 112154).

[0452] Accordingly, methods and uses involving incorporating the microbial (e.g., probiotic) compositions or postbiotic compositions disclosed herein as an adjunct for food or beverage or supplement preservation are envisaged. Effective concentrations of PCA for such methods and uses would be understood in the art and can be used for the disclosed methods.

[0453] In some embodiments, feed and / or food compositions can be prepared by combining a formulated yeast strain with typical animal feed and / or food or drink ingredients. A formulated yeast strain can be used for the preparation of animal feed or food products or beverages, and / or may be added to drinking and / or rearing water. In other embodiments, the compositions of the present invention are feed, food and / or drink additives that are added to a subject’s feed, food, drinking water or beverage prior to ingestion.

[0454] As used herein, “animal feed” includes any animal feed blend known in the art, including rapeseed meal, cottonseed meal, soybean meal, cornmeal, barley, wheat, silage, and haylage.

[0455] The yeast strain disclosed herein can be formulated as a food composition such as a dietary supplement, a functional food, a medical food or a nutritional product as long as the required effect is achieved, i.e. altering or maintaining biological activity in a subject. Said food compositions may be chosen from the group consisting of beverages, yogurts, juices, ice creams, breads, biscuits, crackers, cereals, health bars, spreads, and nutritional products. The food composition may further comprise a carrier material, wherein said carrier material is chosen from the group consisting of lactic acid fermented foods, fermented dairy products, resistant starch, dietary fibers, carbohydrates, proteins and glycosylated proteins. In some embodiments, the yeast strain composition disclosed herein is formulated as a liquid formulation or a solid formulation. When the yeast strain composition (e.g., microbial composition) is a solid formulation, it may be formulated as a tablet, a sucking tablet, a chewing tablet, a chewing gum, a capsule, a sachet, a powder, a granule, a coated particle, a coated tablet, an enterocoated tablet, an enterocoated capsule, a melting strip, or a film. When the microbial 4)

[0456] composition is a liquid formulation, it may be formulated as an oral solution, a suspension, an emulsion or syrup. The composition may further comprise a carrier material independently selected from, but not limited to, the group consisting of vegetables, lactic acid fermented foods, fermented dairy products, resistant starch, dietary fibers, carbohydrates, proteins, and glycosylated proteins.

[0457] PCA can be present in any formulated composition disclosed herein at a concentration depicted in any one of Tables 9-11.

[0458] The Saccharomyces cerevisiae var. boulardii cells and / or cell constituents thereof provided herein can be formulated as a paste (e.g., a cell paste), a powder, dust, a slurry, aqueous or oil-based liquid products, or gel. Common microorganism strain compositions, such as probiotic preparations, are liquid solutions and concentrates or lyophilized powders for resuspension, which can be enclosed in a capsule, vial, or pouch. Such formulations can comprise the engineered S. boulardii yeast strains provided herein and / or cell constituents thereof, in addition to carriers and other agents. As used herein, the term “carrier” refers to an inert compound that is compatible with any other ingredients in the formulation and is not deleterious to the active compound (i.e., engineered yeast cells and / or cell constituents thereof) or a subject that the formulation is administered thereto. Suitable carriers can be added to improve recovery, efficacy, or physical properties and / or to aid in packaging and administration. Such carriers may be added individually or in combination. Nonlimiting examples of carriers include proteins, carbohydrates, fats, enzymes, vitamins, immune modulators, oligosaccharides, milk replacers, minerals, amino acids, coccidiostats, acid-based products, medicines (such as antibiotics), other probiotics, and / or prebiotics. Common carriers include cellulose, sugar, glucose, lactose, whey powder, or rice hulls. The carrier(s) may comprise about 30% weight per weight, weight per volume, or volume per volume, of the final composition. In some embodiments, the carrier(s) may comprise about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98.5%, about 99.0%, about 99.5%, or about 99.9% weight per weight, weight per volume, or volume per volume of the final composition.

[0459] In some embodiments, the yeast strain composition comprises a microbial composition wherein the yeast strains provided herein are formulated as a composition along with a carrier. Such carriers are known in the art and include an inert vehicle, adjuvants, preservatives etc., which are well known. In some embodiments, the carrier comprises one that is not naturally-occurring (i.e., not found in nature). In particular embodiments, the carrier is a naturally- occurring carrier that is not found with a yeast 4)

[0460] strain in the native environment. Such microbial compositions can be prepared in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (21st ed. 2005).

[0461] Any topical formulation of the microbial composition or postbiotic composition, including, but not limited to, a paste (e.g., a cell paste), a powder, dust, a slurry, aqueous or oil-based liquid products, or gel can comprise can comprise an engineered S. boulardii strain disclosed herein at a concentration of at least about 104CFU / g to about 1012CFU / g, about 105CFU / g to about 1012CFU / g, about 106CFU / g to about 1012CFU / g, about 107CFU / g to about 1012CFU / g, about 108CFU / g to about 1012CFU / g, about 109CFU / g to about 1012CFU / g, about 1010CFU / g to about 1012CFU / g, about 1011CFU / g to about 1012CFU / g, about 105CFU / g to about 1011CFU / g, about 105CFU / g to about 1011CFU / g, about 106CFU / g to about 1011CFU / g, about 107CFU / g to about 1011CFU / g, about 108CFU / g to about 1011CFU / g, about 109CFU / g to about 1011CFU / g, about 1010CFU / g to about 1011CFU / g, about 105CFU / g to about 1010CFU / g, about 106CFU / g to about 1010CFU / g, about 107CFU / g to about IO10CFU / g, about 108CFU / g to about 1010CFU / g, or about 109CFU / g to about 1010CFU / g. In other embodiments, the concentration of engineered S. boulardii in a formulation for topical administration comprises or consists of at least about 104CFU / g, at least about 105CFU / g, at least about 106CFU / g, at least about 107CFU / g, at least about 108CFU / g, at least about 109CFU / g, at least about 1010CFU / g, at least about 1011CFU / g, or at least about 1012CFU / g. Another such food or beverage product can comprise a concentration of engineered S. boulardii of at least about 104CFU / ml to about 1012CFU / ml, about 105CFU / ml to about 1012CFU / ml, about 106CFU / ml to about

[0462] 1012CFU / ml, about 107CFU / ml to about 1012CFU / ml, about 108CFU / ml to about 1012CFU / ml, about 109CFU / ml to about 1012CFU / ml, about 1010CFU / ml to about 1012CFU / ml, about

[0463] 1011CFU / ml to about 1012CFU / ml, about 105CFU / ml to about 1011CFU / ml, about 105CFU / ml to about 1011CFU / ml, about 106CFU / ml to about 1011CFU / ml, about 107CFU / ml to about

[0464] 1011CFU / ml, about 108CFU / ml to about 1011CFU / ml, about 109CFU / ml to about 1011CFU / ml, about 1010CFU / ml to about 1011CFU / ml, about 105CFU / ml to about 1010CFU / ml, about 106CFU / ml to about 1010CFU / ml, about 107CFU / ml to about 1010CFU / ml, about 108CFU / ml to about

[0465] 1010CFU / ml, or about 109CFU / ml to about 1010CFU / ml. In other embodiments, the concentration of S. boulardii comprises or consists of at least about 104CFU / ml, at least about 105CFU / ml, at least about 106CFU / ml, at least about 107CFU / ml, at least about 108CFU / ml, at least about 109CFU / ml, at least about 1010CFU / ml, at least about 1011CFU / ml, or at least about 1012CFU / ml.

[0466] Formulations for topical administration can further comprise PCA in any concentration disclosed herein. In some instances, PCA is at least 1%, at least 5%, at least 10%, at least 15%, at least 4)

[0467] 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%. at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% weight per volume (w / v), or volume per volume (v / v), of the formulation, in some instances, PCA is present in the formulation intended for topical administration at a concentration of at least 0.0005 mM (i.e., 0.5 pM), 0.001 mM (i.e., 1 uM), at least 0.005 mM, at least 0.01 mM, at least 0.03 mM, at least 0.06 mM, at least 0.12 mM, at least 0.25 mM, at least 0.5 mM, at least 1 mM, at least 2 mM, at least 5 mM or more than 5 mM. In certain instances, PCA is present at a concentration of between about 0.5 μM to about 5 μM (i.e, 0.005 mM), about 1 μM to about 10 μM (i.e., 0.01 mM), about 0.005 mM to about 0.03 mM, about 0.01 mM to about 0.06 mM, about 0.03 mM to about 0.12 mM, or about 0.06 mM to about 0.2.5 mM.

[0468] PCA can be present in the formulation at a concentration of between about 0.001 mM to about 0.01 mM, about 0.002 mM to about 0.01 mM, about 0.003 mM to about 0.01 mM, about 0.004 to about 0.01 mM, about 0.005 mM to about 0.01 mM, about 0.006 mM to about 0.01 mM, about 0.007 mM to about 0.01 mM, about 0.008 mM to about 0.01 mM, or about 0.009 mM to about 0.01 mM. In other instances, PCA is present at a concentration of between about 0.01 mM to about 0.03 mM, about 0.011 mM to about 0.03 mM, about 0.012 mM to about 0.03 mM, about 0.013 mM to about 0.03 mM, about 0.014 mM to about 0.03 mM, about 0.015 mM to about 0.03 mM, about 0.016 mM to about 0.03 mM, about 0.017 mM to about 0.03 mM about 0.018 mM to about 0.03 mM, about 0.019 mM to about 0.03 mM, about 0.02 mM to about 0.03 mM, about 0.022 mM to about 0.03 mM, about 0.023 mM to about 0.03 mM, about 0.024 mM to about 0.03 mM, about 0.025 mM to about 0.03 mM, about 0.026 mM to about 0.03 mM, about 0.027 mM to about 0.03 mM, about 0.028 mM to about 0.03 mM, about 0.029 mM to about 0.03 mM. In some instances, PCA is present at a concentration of about 0.12 mM. In some instances, PCA is present at a concentration of about 0.03 mM In some instances, PCA is present at a concentration of about 0.01 mM. In some instances, PCA is present at a concentration of about 0.005 mM

[0469] Compositions disclosed herein may also include prebiotics, which may be combined or mixed with a formulated Saccharomyces cerevisiae var. boulardii strain and / or cell constituents thereof into a feed or food composition, into drinking water, or into a microbial composition. Prebiotics are food ingredients that are not readily digestible by enzymes endogenous to the gut (such as those expressed by the animal or those expressed by the resident gut microbiome) and that selectively stimulate the growth and activity of selected groups of intestinal microorganisms that confer beneficial effects upon their host. Typically, it is beneficial microorganism populations that benefit from the presence of 4)

[0470] prebiotic compounds. Prebiotics can consist of oligosaccharides and other small molecules that serve as metabolic substrates for growth of beneficial microbes. Common prebiotics include galactooligosaccharides, fructo-oligosaccharides, inulin, isomalto-oligosaccharies, gentio- oligosaccharides, lactilol, lactosucrose, lactulose, xylosucrose, glycosylsucrose, pyrodextrins, soybean oligosaccharides, guar gum, locust bean gum, arabinan, galactan, pectins, and pectic polysaccharides. While many diverse microbes inhabit the intestinal tract of a host organism, prebiotic compounds are only utilized by the beneficial microbes and lead to a selective enhancement of the...

Claims

CLAIMSWhat is claimed is:

1. A host cell comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ,wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1.

2. The host cell of claim 1, wherein the AroZ or variant of AroZ comprises the amino acid sequence of SEQ ID NO: 1.

3. The host cell of claim 1 or 2, further comprising at least a second heterologous polynucleotide sequence encoding each of the following genes: arol, aro3, aro4, tkll, and tall.

4. The host cell of any one of claims 1-3, wherein the host cell comprises at least two copies of the following genes: arol and aro3.

5. The host cell of any one of claims 1-4, wherein the one or more of the heterologous polynucleotides are codon-optimized.

6. The host cell of any one of claims 1-5, wherein the host cell is a yeast cell.

7. The host cell of claim 6, wherein the yeast cell is a Saccharomyces cerevisiae cell or a Saccharomyces cerevisiae var. boulardii cell.

8. The host cell of any one of claims 1-7, wherein the host cell is a probiotic strain.

9. The host cell of claim 8, wherein the probiotic strain is Bacillus, Bacillus coagulans, Bacillus subtilis, Bifidobacterium, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidus regularis, Bifidobacterium bifidum. Bifidobacterium breve, Bifidobacte ium infarrtis. Bifidobacterium lactis, Bifidobacterium longum, Bifantis, Enterococcus, Escherichia, Escherichia colt, Lacticaseibcicillus, Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, Lactipkintibacillus plantarum. Lactobacillus, Lactobacillus acidophilus, L. brevis, Lactobacilluscasei rhamnosus, Lactobacillus easel, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, L. fermentum, L. gasseri, L. paracesei, L. reuteri, Ligilactobacillus, Ligilaclobadllus salivarius, Limosilactobacillus, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Limosilactobacillus reuteri, Pediococcus, Saccharomyces, Saccharomyces cerevisiae var. boulardii, Streptococcus, or Streptococcus thermophilus.

10. The host cell of claim 8 or 9, wherein the probiotic strain is Saccharomyces cerevisiae var. boulardii.

11. A Saccharomyces cerevisiae var. boulardii host cell comprising a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) comprising an amino acid sequence set forth as SEQ ID NO: 1,wherein said host cell further comprises the following heterologous polynucleotides encoding codon-optimized variants:i) a second heterologous polynucleotide encoding an Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 3;ii) a third heterologous polynucleotide encoding a second Arol enzyme comprising an amino acid sequence set forth as SEQ ID NO: 11;iii) a fourth heterologous polynucleotide encoding a first Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 4;iv) a fifth heterologous polynucleotide encoding a second Aro3 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 12;v) a sixth heterologous polynucleotide encoding an Aro4 enzyme comprising an amino acid sequence set forth as SEQ ID NO: 5;vi) a seventh heterologous polynucleotide encoding a Tkll enzyme comprising an amino acid sequence set forth as SEQ ID NO: 6; andvii) an eighth heterologous polynucleotide encoding a Tall enzyme comprising an amino acid sequence set forth as SEQ ID NO: 7.

12. A microbial composition comprising at least a first host cell, and / or cell constituent(s) thereof,wherein said at least first host cell comprises the host cell of any one of claims 1-11.

13. A microbial composition comprising a plurality of Saccharomyces cerevisiae var. boulardii (S. boulardii) and / or cell constituent(s) thereof,wherein said S. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ,wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of SEQ ID NO: 1.

14. The microbial composition of claim 12 or 13, wherein said plurality comprises about 104CFU / gram to about 1012CFU / gram or about 104CFU / ml to about 1012CFU / ml of 5. boulardii.

15. The microbial composition of any one of claims 12-14, further comprising protocatechuic acid (PCA).

16. The microbial composition of claim 15, wherein said PCA is present at a concentration of at least 0.0001 mM (i.e., 0.1 pM), at least 0.0005 mM, 0.001 mM (i.e., 1 pM), at least 0.005 mM, at least 0.01 mM, at least 0.03 mM, at least 0.06 mM, at least 0.12 mM, at least 0.25 mM, at least 0.5 mM, at least 1 mM, at least 2 mM.

17. The microbial composition of claim 16, wherein said PCA is present at a concentration of between about 0.005 mM (i.e., 5 pM) to about 0.06 mM.

18. The microbial composition of any one of claims 12-17, further comprising i) luminal contents of the gastrointestinal tract of a subject; ii) at least one intestinal epithelial cell; and / or iii) at least one skin epithelial cell.

19. A Saccharomyces cerevisiae var. boulardii (S. bou / ardiij-denved postbiotic composition, said composition comprising a plurality of S. boulardii, and / or cell constituent(s) thereof, wherein said S. boulardii comprises a heterologous polynucleotide encoding a Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ,wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1, andwherein 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%, or at least 99% of said plurality is non-viable.

20. The postbiotic composition of claim 19, wherein said plurality comprises no more than:i) 1000 CFU / gram, 100 CFU / gram, 10 CFU / gram, or 1 CFU / gram; and / orii) 1000 CFU / ml, 100 CFU / ml, 10 CFU / ml, or 1 CFU / ml,of said S. boulardii.

21. The postbiotic composition of claim 19 or 20, further comprising protocatechuic acid (PCA).

22. The postbiotic composition of claim 21, wherein said PCA is present at a concentration of at least 0.0001 mM (i.e., 0.1 pM), at least 0.0005 mM, 0.001 mM (i.e., 1 pM), at least 0.005 mM, at least 0.01 mM, at least 0.03 mM, at least 0.06 mM, at least 0.12 mM, at least 0.25 mM, at least 0.5 mM, at least 1 mM, at least 2 mM.

23. The postbiotic composition of claim 22, wherein said PCA is present at a concentration of between about 0.005 mM (i.e., 5 pM) to about 0.06 mM.

24. The postbiotic composition of any one of claims 19-23, further comprising i) luminal contents of the gastrointestinal tract of a subject; ii) at least one intestinal epithelial cell; and / or iii) at least one skin epithelial cell.

25. The microbial composition of any one of claims 12-18 or the postbiotic composition of any one of claims 19-24, wherein said cell constituent(s) comprises at least one postbiotic agent.

26. The microbial composition or postbiotic composition of claim 25, wherein said at least one postbiotic agent comprises a lipid, a carbohydrate, a lipoprotein, a glycolipid, a glycoprotein, a metabolite, genetic material, or any combination thereof.

27. The microbial composition of any one of claims 12-18, 25, or 26 or the postbiotic composition of any one of claims 19-26, further comprising a prebiotic.

28. The microbial composition of any one of claims 12-18 or 25-27 or the postbiotic composition of any one of claims 19-27, wherein said composition is formulated as a capsule, gel, softgel, hydrogel, paste, tablet, gummy, spray, aerosol, lozenge, effervescent tablet, orodispersible film, powder, or liquid.

29. The microbial composition or postbiotic composition of claim 28, wherein the formulation is a liquid formulation.

30. The microbial composition or postbiotic composition of claim 28, wherein the formulation is a solid formulation.

31. The microbial composition of any one of claims 12-18 or 25-30 or the postbiotic composition of any one of claims 19-30, further comprises a nutritional supplement.

32. The microbial composition of any one of claims 12-18 or 25-31 or the postbiotic composition of any one of claims 19-31, wherein said at least first microorganism is:i) lyophilized;ii) pasteurized; and / oriii) freeze-dried.

33. A food or beverage product comprising said microbial composition of any one of claims 12-18 or 25-32 or the postbiotic composition of any one of claims 19-32.

34. The food or beverage product of claim 33, wherein said product is a nutritional whole food, drink, sports drink, coffee, tea, water, mineral water, soda, carbonated beverage, oral hydration drink, mineral water, soup, replacement food, nutritional bar, nutritional consumable, gummy, tablet, confectionery, fermented or unfermented milk-based product, yogurt product, milk-based powder, enteral nutritional product, fermented or unfermented cereal-based product, chocolate, milk, yogurt, cheese, ice cream, baby and infant formula, cereal, animal feed, protein shake, protein powder, functional waterjuice blend, smoothie, ready-to-drink (RTD) beverage, non-dairy milk, nut or seed butter, energy drink, functional shot, baked good, granola bar, snack bar, alcoholic beverage, frozen food, frozen meal, condiment, pasta, noodles, pet food, or pet treat.

35. A method of altering or maintaining biological activity in a subject in need thereof, said method comprising administering to said subject an effective amount of the microbial composition of any one of claims 12-18 or 25-32, the postbiotic composition of any one of claims 19-32, or the food or beverage product of claim 33 or 34.

36. The method of claim 35, wherein said altered or maintained biological activity comprises any one of the following:i. suppressing expression of at least one gene associated with inflammation in at least one cell present in said subject;ii. stimulating an epithelial tissue response in said subject;iii. reducing at least one symptom of a gastrointestinal or skin disorder in said subject; iv. increasing levels of protocatechuic acid (PCA) in at least one tissue in said subject;and / orv. improving well-being of said subject,compared to a control, wherein said control comprises a subject who is not administered an effective amount of said composition.

37. The method of claim 36, wherein said at least one gene is iNOS and / or COX-2.

38. The method of any one of claims 35-37, wherein said effective amount of the microbial composition comprises about 105CFU / gram to about 1012CFU / gram or about 105CFU / ml to about 1012CFU / ml of said S. boulardii.

39. The method of any one of claims 35-38, wherein said composition is administered to the gastrointestinal tract of the subject.

40. A method of producing a Saccharomyces cerevisiae var. boulardii (S. bou / ardiij-denved postbiotic composition comprising a plurality of S. boulardii, and / or cell constituent(s) thereof, said method comprisinginoculating at least one S. boulardii cell into a cell culture media,incubating said inoculated culture medium under conditions suitable for growth and proliferation of said S. boulardii, andisolating a cell-free cell supernatant from said culture medium,thereby producing said postbiotic composition,wherein said S. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ, wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1, andwherein at least 90%, at least 95%, or at least 99% of said plurality is non-viable.

41. The method of claim 40, wherein said incubation involves a fermentation step.

42. The method of claim 40 or 41, wherein the number of viable S. boulardii cells are reduced.

43. The method of any one of claims 40-42, wherein said postbiotic composition is:i) lyophilized;ii) pasteurized; and / oriii) freeze-dried.

44. The method of any one of claims 40-43, wherein said method further comprises a step of formulating a food or beverage product comprising said postbiotic composition.

45. A method of producing protocatechuic acid (PC A) in a microbial culture, said method comprisinginoculating at least one cell of an engineered Saccharomyces cerevisiae var boulardii (S. boulardii) strain capable of producing PCA in a culture media, andincubating said medium under conditions suitable for growth and proliferation of said S. boulardii,whereby PCA is secreted into said medium,thereby producing a composition comprising PCA.

46. The method of claim 45, wherein said PCA is present at a concentration of at least 12 g / L.

47. The method of claim 45 or 46, wherein said method further involves converting PCA into a second molecule.

48. The method of claim 47, wherein PCA is a precursor for said second molecule.

49. The method of claim 47, wherein said second molecule comprises vanillin, muconic acid, and / or adipic acid.

50. The method of any one of claims 45-48, comprising purifying said PCA or said second molecule.

51. The method of any one of claims 45-50, wherein said 5. boulardii comprises a heterologous polynucleotide encoding an Aspergillus oryzae 3-dehydroshikimate dehydratase (AroZ) or a variant of AroZ,wherein the AroZ or the variant of AroZ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, or a variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, or a conservatively substituted version of the amino acid sequence set forth as SEQ ID NO: 1.

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