Engineered saccharomyces and uses thereof

Abstract

Provided herein are engineered yeast having a heterologous nucleic acid genomically integrated into one or more safe harbor sites. Methods of using the engineered yeast for therapeutic and non-therapeutic utilities are also provided. Compositions comprising engineered yeast and methods of using said engineered yeast for therapeutic and non-therapeutic utilities are also provided. Methods to make the disclosed engineered yeast are also provided.

Description

Attorney Matter No. TNZ-017WO ENGINEERED SACCHAROMYCES AND USES THEREOF

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 686,964 filed August 26, 2024, which is hereby incorporated by reference in its entirety. Field

[0002] The present disclosure relates generally to engineered yeast having a heterologous nucleic acid genomically integrated into one or more safe harbor sites for increased secretion of a recombinant protein. Background

[0003] Production of heterologous proteins in probiotic yeasts such as Saccharomyces boulardii requires the generation of a strain with stable and reliable expression of the recombinant protein. Plasmids used to express recombinant proteins require the use of antibiotics for their maintenance, are inherently unstable and are incompatible with biocontainment requisites for a probiotic strain.

[0004] Consequently, an expression system that is to be used in a probiotic strain should be integrated into the genome and exhibit optimal expression level of the desired protein, thus necessitating integration of multiple copies of an expression cassette into the genome.

[0005] A need therefore exists for an engineered yeast system that not only increases the secretion of a recombinant protein produced in yeast, but has a recombinant protein that is genomically integrated at one or more sites and is not dependent on the use of a selection marker for reliable expression. Summary

[0006] In one aspect, provided herein, is an engineered yeast cell comprising a heterologous nucleic acid genomically integrated into one or more safe harbor sites.

[0007] In some embodiments, the yeast cell is a yeast from the genus Saccharomyces. In some embodiments, the yeast cell is a Saccharomyces species selected from the group comprising Saccharomyces cerevisiae and Saccharomyces boulardii. In some embodiments, the yeast cell is the Saccharomyces species Saccharomyces boulardii.

[0008] In some embodiments, the heterologous nucleic acid is integrated at about 3 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, about 100 or more, about 105 or more, about 110 or more, about 115 or more, about 120 or more, about 125 or more, about 130 or more, about 135 1 IPTS / 200107384.1Attorney Matter No. TNZ-017WO or more, about 140 or more, about 145 or more, or about 150 or more safe harbor sites. In some embodiments, the heterologous nucleic acid is integrated at about 3 or more, about 5 or more, about 10 or more, about 15 or more, or about 20 or more safe harbor sites. In some embodiments, the heterologous nucleic acid is integrated at about 10 or more or about 15 or more safe harbor sites. In some embodiments, the heterologous nucleic acid is integrated at about 14 safe harbor sites.

[0009] In some embodiments, the safe harbor sites include one or more safe harbor sites selected from the group comprising a long terminal repeat (LTR) of a transposable element (Ty element), a non-essential gene, a metabolic auxotrophic gene, and a transcriptionally inactive region in the genome. In some embodiments, the safe harbor sites include one or more safe harbor sites from a long terminal repeat (LTR) of a Ty element and a metabolic auxotrophic gene. In some embodiments, the one or more metabolic auxotrophic genes are selected from the group comprising LEU2 (YCL018W), HIS3 (YOR202W), and URA3 (YEL021W).

[0010] In some embodiments, the metabolic auxotrophic gene comprises a 5’ insertion and a 3’ insertion point.

[0011] In some embodiments, the 5’ insertion and a 3’ insertion point are selected from the group comprising the pairs 1-3: Pair Sequence 5’ of insertion point Sequence 3’ of insertion point AT A CT 6) [, y ed in chromosome number 1, 2, 4, 5, 6, 7, 13, 14, 15, or 16 of Saccharomyces boulardii.

[0013] In some embodiments, each of the one or more LTRs of a Ty element comprise a 5’ insertion and a 3’ insertion point.

[0014] In some embodiments, the 5’ insertion and a 3’ insertion point are selected from the group comprising the pairs 4-15: Pair Sequence 5’ of insertion point Sequence 3’ of insertion point TT 9)2 IPTS / 200107384.1Attorney Matter No. TNZ-017WO Pair Sequence 5’ of insertion point Sequence 3’ of insertion point 5 ATTATGTATACAGAATATACTTTAGAAGTTC ATAATGAAACATTTAAAACGGAATGAGGA TCCTCGATGATATAGGAAT (SEQ ID NO 8) ATAATCGTAATATTATTATGT (SEQ ID NO: A O: TC T AT : AG 5) T TC C O: T O: C :quence that encodes a payload protein.

[0016] In some embodiments, the payload protein is an enzyme, a growth factor, insulin, an incretin, a cytokine, an antibody or fragment thereof, an antimicrobial peptide, a mucosal protein, an agricultural product, a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, a nutritional protein, an antiviral, an enzyme inhibitor, or a hormone.

[0017] In some embodiments, the enzyme is selected from the group comprising invertase, isomaltase, lactase, lysozyme, and An-PEP. In some embodiments, the enzyme is invertase.

[0018] In some embodiments, the incretin is selected from the group comprising GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, and nesfatin.

[0019] In some embodiments, the mucosal protein is selected from the group comprising trefoil factor, Reg3 protein, and superoxide dismutase.

[0020] In some embodiments, the agricultural product is selected from the group comprising pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, and fertilizer. 3 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0021] In some embodiments, the heterologous nucleic acid comprises a signal peptide. In some embodiments, the signal peptide is as provided for in WO2022 / 192675. In some embodiments, the signal peptide comprises a pre-protein signal peptide, a pro-protein signal peptide, or a combination thereof. In some embodiments, the pre-protein signal peptide is selected from: MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32), or MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33). In some embodiments, the pro-protein signal peptide is selected from: EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), or DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36). In some embodiments, the signal peptide comprises: MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), or MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36). In some embodiments, the signal peptide comprises the sequence of MKFKLTLLAALLALAALVLAASEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASR HKRDV (SEQ ID NO: 37).

[0022] In some embodiments, the heterologous nucleic acid comprises a homology arm at the 5’ end and a homology arm at the 3’ end of the heterologous nucleic acid. In some embodiments, the 4 IPTS / 200107384.1Attorney Matter No. TNZ-017WO homology arm at the 5’ end and the homology arm at the 3’ end of the heterologous nucleic acid comprise a sequence complementary to the 5’ insertion site and 3’ insertion stie, respectively.

[0023] In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-3 and 7-18. In some embodiments, the homology arm at the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4-6 and 19- 30.

[0024] In some embodiments, the heterologous nucleic acid comprises a constitutive or inducible promoter. In some embodiments, the constitutive or inducible promoter is a TEF1 promoter.

[0025] In some embodiments, the heterologous nucleic acid comprises one or two terminator sequences. In some embodiments, the one or two terminator sequences are a CYC1 terminator or a ADH1 terminator.

[0026] In some embodiments, the heterologous nucleic acid is integrated at 14 safe harbor sites and comprises a nucleic acid sequence that encodes invertase.

[0027] In some embodiments, the heterologous nucleic acid comprises the following elements operably linked from 5’ to 3’: a CYC1 terminator sequence, a TEF1 promoter sequence, a Sb propeptide sequence, a sequence that encodes invertase, and a ADH1 terminator sequence.

[0028] In some embodiments, the engineered yeast cell further comprises a nuclease capable of generating a break in the 5’ integration site or 3’ integration site.

[0029] In some embodiments, the nuclease is a nuclease selected from the group comprising TALEN, ZFN, a Cas protein, and HO endonuclease. In some embodiments, the nuclease is a Cas protein. In some embodiments, the nuclease is Cas9.

[0030] In another aspect ,provided herein is a method of making the engineered yeast cell disclosed herein comprising targeted insertion of a heterologous nucleic acid into one or more safe harbor sites.

[0031] In some embodiments, the one or more safe harbor sites each comprise a 5’ integration site and a 3’ integration site.

[0032] In some embodiments, the method comprises a nuclease capable of generating a break in the 5’ integration site or 3’ integration site. In some embodiments, the nuclease is a nuclease selected from the group comprising TALEN, ZFN, a Cas protein, and HO endonuclease. In some embodiments, the nuclease is a Cas protein. In some embodiments, the nuclease is Cas9.

[0033] In another aspect, provided herein is a method for treating a disease or a condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the engineered yeast cell disclosed herein. 5 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0034] In some embodiments, the disease or condition is selected from an infection, an autoimmune disease, primary (congenital) enzymatic deficiency, enzymatic deficiencies secondary to functional gut disorders, diabetes, obesity, a metabolic disorder, intestinal bacterial overgrowth, enteric infection, bacterial vaginosis, inflammatory bowel disease, irritable bowel syndrome, small bowel syndrome, Celiac disease, gluten intolerance, colitis, peptic ulcer, or another GI condition or disorder.

[0035] In some embodiments, the disease or condition is an enzyme deficiency and the payload protein is an enzyme.

[0036] In some embodiments, the disease or condition is congenital sucrase-isomaltase deficiency and the payload protein is one or both of invertase and isomaltase.

[0037] In some embodiments, the disease or condition is one or both of sucrose and isomaltase intolerance secondary to a functional gut disorder and the payload protein is one or both of invertase and isomaltase.

[0038] In some embodiments, the disease or condition is one or more of gluten intolerance, refractory sprue, or Celiac disease and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide.

[0039] In some embodiments, the disease or condition is pancreatitis or exocrine pancreatic insufficiency and the payload protein is selected from one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin.

[0040] In some embodiments, the disease or condition is enteropeptidase deficiency or enterokinase deficiency and the payload protein is one or all of enteropeptidase, proenteropeptidase, and enterokinase.

[0041] In some embodiments, the disease or condition is small intestinal bacterial overgrowth, inflammatory bowel disease, irritable bowel syndrome, C. difficile infection, cystic fibrosis, necrotizing enterocolitis, and diabetes, and the payload protein is intestinal alkaline phosphatase.

[0042] In some embodiments, the disease or condition is short bowel syndrome and the payload protein is IGF-1, GLP-2, or a synthetic derivative of GLP-2.

[0043] In some embodiments, the disease or condition is lactose sensitivity or lactose intolerance and the payload protein is lactase.

[0044] In some embodiments, the disease or condition is trehalose sensitivity or lactose intolerance and the payload protein is trehalase.

[0045] In some embodiments, the disease or condition is maltose sensitivity or lactose intolerance and the payload protein is maltase.

[0046] In some embodiments, the disease or condition is pernicious anemia and the payload protein is intrinsic factor. 6 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0047] In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is lysozyme, nisin, a defensin, magainin, cateslytin, or any combination thereof.

[0048] In some embodiments, the condition is a bacterial infection caused by one or more of E. coli, C. difficile, vibrio cholera, Shigella, Salmonella, Cryptosporidium, or any combination thereof.

[0049] In some embodiments, the condition is a viral infection.

[0050] In some embodiments, the disease or condition is type 1 or type 2 diabetes mellitus and the payload protein is insulin, or an incretin.

[0051] In some embodiments, the administration is oral or topical.

[0052] In some embodiments, the disease or condition has an inflammatory component and the payload protein is IL-10, IL-22, TGFβ, an anti-TNFα antibody or fragment thereof, or any combination thereof.

[0053] In some embodiments, a method for treating a disease or condition in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a yeast as provided for herein. Description of the Drawings

[0054] The foregoing and other features of the disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

[0055] FIG.1 shows an exemplary heterologous nucleic acid construct containing a sequence that encodes invertase.

[0056] FIG.2A, 2B, and 2C show a summary of the construction of engineered S. boulardii strains in which an exemplary invertase construct was genomically integrated into safe harbor sites. FIG.2A illustrates the safe harbor sites utilized, and the integration sites utilized for each strain (2X, 4X, etc.) are indicated with a checkmark. FIG.2B and 2C illustrate how a yeast cell with invertase genomically integrated may be used as a template to insert unique genes in place of the invertase. FIG.2B illustrates the exemplary nucleic acid construct of FIG.1 with invertase specific gRNA sites indicated. FIG.2C illustrates the safe harbor sites and invertase specific gRNAs that may be utilized to insert new genes in place of the invertase genes for each site.

[0057] FIG.3 shows the invertase activity of the engineered S. boulardii strains with the indicated number of copies of invertase integrations. 7 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0058] FIG.4 shows the invertase activity an engineered S. boulardii strains with 28 of copies of invertase genomic integrations compared to a wild-type S. boulardii strain and a S. boulardii strain with a plasmid that encodes invertase.

[0059] FIG.5A, 5B, 5C, and 5D shows exemplary heterologous nucleic acid constructs containing a sequence that encode a payload protein, human interleukin-10 (hIL-10), with a pre- and pro-protein signal peptide. “Sb v1” comprises or consists of SEQ ID NO: 31, “Sb v2” comprises or consists of SEQ ID NO: 61, “Sb v3” comprises or consists of SEQ ID NO: 62; “Sb v4” comprises or consists of SEQ ID NO: 63, “Sb v5” comprises or consists of SEQ ID NO: 64; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34. FIG.5A illustrates exemplary nucleic acid constructs wherein the IL-10 dimer remains constant and the pre-peptide, pro- peptide, or promoter sequences are varied. FIG.5B, 5C, and 5D illustrate exemplary nucleic acid constructs wherein the pre-peptide, pro-peptide, and promoter sequences remain constant and the IL-10 dimer sequence is varied based on different linker compositions.

[0060] FIG.6 shows exemplary heterologous nucleic acid constructs containing a sequence that encode a payload protein, human interleukin-21 (hIL-21), with a pre- and pro-protein signal peptide. “Sb v1” comprises or consists of SEQ ID NO: 31, “Sb v2” comprises or consists of SEQ ID NO: 61, “Sb v3” comprises or consists of SEQ ID NO: 62; “Sb v4” comprises or consists of SEQ ID NO: 63, “Sb v5” comprises or consists of SEQ ID NO: 64; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34.

[0061] FIG.7 shows exemplary heterologous nucleic acid constructs containing a sequence that encode a payload protein, mouse interleukin-22 (mIL-22), with a pre- and pro-protein signal peptide. “Sb v1” comprises or consists of SEQ ID NO: 31, “Sb v2” comprises or consists of SEQ ID NO: 61, “Sb v3” comprises or consists of SEQ ID NO: 62; “Sb v4” comprises or consists of SEQ ID NO: 63, “Sb v5” comprises or consists of SEQ ID NO: 64; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34.

[0062] FIG.8 shows exemplary heterologous nucleic acid constructs containing a sequence that encode a payload protein, human interleukin-22 (hIL-22), with a pre- and pro-protein signal peptide. “Sb v1” comprises or consists of SEQ ID NO: 31, “Sb v2” comprises or consists of SEQ ID NO: 61, “Sb v3” comprises or consists of SEQ ID NO: 62; “Sb v4” comprises or consists of SEQ ID NO: 63, “Sb v5” comprises or consists of SEQ ID NO: 64; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34.

[0063] FIG.9 shows exemplary heterologous nucleic acid constructs containing a sequence that encode a payload protein, human interleukin-2 (hIL-2), with a pre- and pro-protein signal peptide. “Sb v1” comprises or consists of SEQ ID NO: 31; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34. 8 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0064] FIG.10 shows exemplary heterologous nucleic acid constructs containing a sequence that encode a payload protein, with a pre- and pro-protein signal peptide. Payload proteins include human interleukin-10 (hIL-10) mutants, expressed either singly or as a dimer; hIL-22, or both hIL-10 and h-IL22. “Sb v1” comprises or consists of SEQ ID NO: 31; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34. For dual construct 1 (dual#1) the construct is broken up only for ease of visualization. The construct runs continuously through point “A” in the figure, such that the 3’ end of the construct in line 1 ending in “A” is connected to the 5’ end of the construct in the line below beginning with “A”. The same is true for dual construct 2 (dual#2), wherein “B” is used to indicate the connection point.

[0065] FIG.11A, 11B, and 11C show exemplary heterologous nucleic acid constructs containing a sequence that encode a payload protein, human intestinal alkaline phosphatase (hIAP), with a pre-protein signal peptide and with and without a pro-protein signal peptide. “Sb v1” comprises or consists of SEQ ID NO: 31, “Sb v2” comprises or consists of SEQ ID NO: 61, “Sb v5” comprises or consists of SEQ ID NO: 64; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34. FIG.11A illustrates constructs containing only the hIAP with pre-protein signal peptide sequences and with or without pro-protein signal peptide sequences. FIG.11B and 11C illustrate the same constructs as in FIG.11A with the addition of anchoring proteins Sed1 or Cwp2.

[0066] FIG.12A, 12B, and 12C show exemplary heterologous nucleic acid constructs containing a sequence that encode a payload protein, yeast alkaline phosphatase (yAP), with a pre-protein signal peptide and with and without a pro-protein signal peptide. “Sb v1” comprises or consists of SEQ ID NO: 31, “Sb v2” comprises or consists of SEQ ID NO: 61, “Sb v5” comprises or consists of SEQ ID NO: 64; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34. FIG. 12A illustrates constructs containing only the yAP with pre-protein signal peptide sequences and with or without pro-protein signal peptide sequences. FIG.12B and 12C illustrate the same constructs as in FIG.12A with the addition of anchoring proteins Sed1 or Cwp2.

[0067] FIG.13A shows an exemplary heterologous nucleic acid constructs containing a sequence that encode a payload protein, yeast alkaline phosphatase (yAP), with a pre-protein signal peptide (Sb v5), a pro-protein signal peptide (Sb propeptide 1), and an anchoring protein (Sed1). This construct is identical to yAP#16 as shown in FIG.12C. .“Sb v5” comprises or consists of SEQ ID NO: 64; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34.

[0068] FIG.13B illustrates the results of alkaline phosphatase activity in yeast cells with yAP genomically integrated at 2X, yAP transiently expressed, or no expression control. In each instance, the yAP construct utilized is yAP#16 as shown in FIG.12C. 9 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0069] FIG.13C illustrates the results of alkaline phosphatase activity in yeast cells with yAP genomically integrated at 2X, yAP transiently expressed, or no expression control. In each instance, the yAP construct utilized is yAP#6 as shown in FIG.12A. Detailed Description

[0070] The present disclosure presents a solution to the aforementioned challenges by providing new, synthetic signal peptides that direct secretion of expressed proteins or peptides in yeast. The disclosed signal peptides overcome performance variability challenges posed by previously characterized and native signal peptides and may be used to generate and facilitate secretion of any protein or peptide from a yeast.

[0071] The disclosed engineered yeast achieve increased secretion of any recombinant protein by having a heterologous nucleic acid genomically integrated into one or more safe harbor sites. In any embodiment, the heterologous nucleic acid may further comprise a synthetic pre-protein signal peptide. Likewise, in any embodiment, the heterologous nucleic acid may further comprise a synthetic pro-protein signal peptide. The use of synthetic pro-protein signal peptide together with a synthetic pre-protein signal peptide may further improve secretion of a payload protein. Without being bound to any particular theory, the engineered yeast disclosed herein provide stable and sustained expression and release of the desired proteins or peptides while not depending on an external regulator, such as an antibiotic, for reliable expression. The necessity of a microorganism to rely on, for example, an antibiotic resistance gene to obtain stable and sustained expression of a desired protein product is a significant barrier in the use of microorganisms for a variety of utilities. The engineered yeast of the present disclosure overcome these problems and others. Use of the disclosed engineered yeast may be used to achieve increased secretion of any desired payload in a yeast compatible environment, such as in therapeutics, agriculture, or food products.

[0072] Before the present compositions and methods are described, it is to be understood that the scope of the invention is not limited to the particular processes, compositions, or methodologies described herein, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the methods and systems disclosed herein, the preferred methods, devices, and materials are now described.

[0073] Definitions 10 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0074] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.

[0075] As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. For example, reference to “comprising a therapeutic agent” includes one or a plurality of such therapeutic agents. The term “or” refers to a single element of stated alternative elements, unless the context clearly indicates otherwise. For example, the phrase “A or B” refers to A alone or B alone. The phrase “A, B, or a combination thereof” refers to A alone, B alone, or a combination of A and B. Similarly, “one or more of A and B” refers to A, B, or a combination of both A and B. The phrase “A and B” refers to a combination of A and B. Furthermore, the various elements, features and steps discussed herein, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in particular examples.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. All references cited herein are incorporated by reference in their entirety.

[0077] In some examples, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments are to be understood as being modified in some instances by the term "about" or "approximately." For example, "about" or "approximately" can indicate + / - 5% variation of the value it describes. Accordingly, in some embodiments, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties for a particular embodiment. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some examples are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.

[0078] To facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided: 11 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0079] As used herein, “yeast” refers to a microscopic fungus consisting of cells that reproduce by budding and are capable of converting sugar into alcohol and carbon dioxide. The yeast, as disclosed herein may be genetically modified to induce expression of a heterologous payload protein. As used herein, “genetically modified” or any grammatical variation thereof, refers to a practice of introducing a nucleic acid or a nucleic acid molecule into a yeast cell that encodes and promotes the expression of a recombinant protein. The nucleic acid may be introduced transiently, or the nucleic acid may be incorporated into the genome of the yeast for stable expression. As used herein, the terms “nucleic acid” and “nucleic acid molecule” can be used interchangeably. The nucleic acid or nucleic acid molecule can be of any length. A nucleic acid may be DNA, mRNA, tRNA, or rRNA. A nucleic acid or nucleic acid molecule is composed of nucleotide monomers, each triplet of monomers (a codon) encoding for either a triplet of RNA nucleotide monomers (if the nucleic acid is DNA) or an amino acid (if the nucleic acid is RNA). DNA also comprises one or more promoter regions, which indicate where transcription of the DNA should start. mRNA also comprises a ribosome binding site, which indicates where translation of the mRNA should start as well as one or more stop codons, which indicates where mRNA translation should end. The introduction of a nucleic acid or nucleic acid molecule into a yeast cell can be accomplished by any method known in the art. Such methods are described in greater detail below.

[0080] In any embodiment or aspect disclosed herein, a nucleic acid encoding for a recombinant polypeptide, as disclosed herein, may be introduced into a yeast cell using any method known to those skilled in the art for such introduction. Such methods include transfection, transformation, transduction, infection (e.g., viral transduction), injection, microinjection, gene gun, nucleofection, nanoparticle bombardment, transformation, conjugation, by application of the nucleic acid in a gel, oil, or cream, by electroporation, using lipid-based transfection reagents, or by any other suitable transfection method. One of skill in the art will readily understand and adapt such methods using readily identifiable literature sources.

[0081] As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection (e.g., using commercially available reagents such as, for example, LIPOFECTIN® (Invitrogen Corp., San Diego, CA), LIPOFECTAMINE® (Invitrogen), FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI™ (Polyplus-transfection Inc., New York, NY), EFFECTENE® (Qiagen, Valencia, CA), DREAMFECT™ (OZ Biosciences, France) and the like), or electroporation (e.g., in vivo electroporation). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., 12 IPTS / 200107384.1Attorney Matter No. TNZ-017WO Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

[0082] Methods and materials of non-viral delivery of nucleic acids to cells further include biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid-nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in U.S. Pat. Nos.5,049,386, 4,946,787; and 4,897,355 and lipofection reagents are sold commercially (e.g., TRANSFECTAM™ and LIPOFECTIN™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in WO91 / 17424 and WO 91 / 16024.

[0083] The methods described herein comprise generating a genetically modified yeast comprising a heterologous nucleic acid molecule encoding for a polypeptide of interest within a yeast host. As used herein, heterologous or recombinant describes a protein or nucleic acid that is not naturally found in or produced by the host yeast. As used herein, a “recombinant polypeptide” comprises a payload protein and a synthetic signal peptide fused directly or indirectly thereto. As used herein, “recombinant polypeptide” and “recombinant fusion protein” may be used interchangeably in the context of polypeptides comprising at least a first and second component (e.g. a synthetic signal peptide and a payload protein) or at least a first, second, and third component (e.g. a synthetic signal peptide, a payload protein, and an anchoring protein). As used herein, a signal peptide is any protein or peptide fused directly or indirectly to the N-terminus of a payload protein that facilitates the extracellular secretion of the payload protein after it is generated. A signal peptide may comprise one or more of a pre-protein signal peptide and pro- protein signal peptide.

[0084] The chemical makeup of a peptide or polypeptide will be described herein by a series of amino acid single letter abbreviations or an “amino acid sequence / s” or “sequence / s,” which are conventional and known to those in the art. While reference sequences will be explicitly disclosed, in any aspect and embodiment, a reference sequence may be modified to include conservative amino acid substitutions, as well as variants and fragments, while maintaining the characteristics and functionality of the reference sequence.

[0085] The methods disclosed herein utilize a synthetic signal peptide to increase extracellular secretion of a payload protein by a yeast. As used herein, a “synthetic signal peptide” refers to a signal peptide whose sequence is generated as provided for herein and that is made recombinantly. The recombinantly produced signal peptide can be referred to as a “synthetic signal peptide” or simply as a “signal peptide”. The signal peptide comprising one or more of a synthetic pre-protein (sPre) signal peptide and a synthetic pro-protein (sPro) signal peptide. As highlighted previously, the term synthetic in this context refers to a recombinantly produced pre-protein signal peptide or 13 IPTS / 200107384.1Attorney Matter No. TNZ-017WO pro-protein signal peptide whose sequence is generated as provided for herein. Hereafter, the pre- and pro-signal peptides may be referred to as “synthetic” pre or pro-protein signal peptides, or simply as pre or pro-protein signal peptides. In embodiments where a native pre or pre-protein signal peptide is utilized or referred to, the peptide will be denoted as such. In the context of this application, the term “native” refers to a pre or pro signal peptide the sequence of which is adopted, in whole or in part, from a known pre or pro signal peptide sequence at the time of this application. In other words, the “native” signal peptides are not generated using the formulas or methods as provided for herein. However, it is to be understood that a synthetic signal peptide may comprise a synthetic pre-protein signal peptide fused with a native pro-protein signal peptide (sPre-nPro signal peptide). In another example, a synthetic signal peptide may comprise a native pre-protein signal peptide fused to a synthetic pro-protein signal peptide (nPre-sPro signal peptide). In yet another example, a synthetic signal peptide comprises a synthetic pre-protein signal peptide and no pro-protein signal peptide. Similarly, a synthetic signal peptide may comprise a synthetic pro- protein signal peptide but no pre-protein signal peptide.

[0086] A pre-protein signal peptide (synthetic or native) comprises 10 to 50 amino acids, which are appended either directly to the N-terminus of a payload protein or indirectly to the N-terminus of a payload protein, with one or more of a Kex protease (KR) site, Ste13 cleavage site, and spacer there between.

[0087] A pro-protein signal peptide comprises 10 to 200 amino acids that are appended either directly to the N-terminus of a payload protein or indirectly to the N-terminus of a payload protein, with one or more of a KR site, Ste13 cleavage site, and spacer there between. Many proteins are natively expressed comprising a pro-protein signal peptide, though, as will be described, these native pro-protein signal peptides often lack the activity to generate sufficient secretion of a payload protein. The various synthetic signal peptides described herein may be used as a replacement of all or part of a native signal peptides.

[0088] A pre- and / or pro-protein signal peptide, whether synthetic or native, may be appended to an adjacent amino acid via a bond to the N-terminal amino acid of the adjacent amino acid, for example, by a peptide bond, a dipeptide spacer, or a membrane-associating / lipidophilic alpha- helical peptide signal peptide (e.g., MISTIC, represented by the amino acid sequence FCTFFEKHHRKWDILLEKSTGVMEA (SEQ ID NO: 79) or SEQ ID NO.26).

[0089] As used herein, “payload protein” or “protein of interest” refers to the protein that will be generated by the host and chaperoned through the secretory pathway into the extracellular space, facilitated by the presence of a synthetic signal peptide. Upon secretion into the extracellular space, all, some, or none of the synthetic signal peptide may be fused to the payload protein. Optionally, a payload protein still being attached partially or fully to the synthetic signal peptide may be further 14 IPTS / 200107384.1Attorney Matter No. TNZ-017WO processed, for example, to remove the remaining signal peptide. A payload protein may be any protein known or yet to be known, for example, an enzyme, enzyme inhibitor, growth factor, cytokine, hormone, antibody, antigen, vaccine, a therapeutic agent, or any combination thereof. More specific examples follow herein below.

[0090] The compositions disclosed herein may be provided to a subject in a variety of ways through administration of the composition to the subject. As used herein, administer or administration means to provide or the providing of a composition to a subject. Oral administration, as used herein, refers to delivery of an active agent through the mouth. Topical administration, as used herein, refers to the delivery of an active agent to a body surface, such as the skin, a mucosal membrane (e.g., nasal membrane, vaginal membrane, buccal membrane, or the like).

[0091] A payload protein secreted by the various genetically modified yeast disclosed herein, which are interchangeably referred to as “engineered yeast”, may be provided to a subject in a pharmaceutical composition. Additionally or alternatively, the engineered yeast itself may be provided to a subject in a pharmaceutical composition.

[0092] The various compositions disclosed herein may be useful in treating a number of diseases or conditions, for example, cancer. As used herein, cancer refers to a condition characterized by unregulated cell growth. Examples of cancer include, but are not limited to, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, cervical cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, and esophageal cancer. In some embodiments, the diseases or conditions may include, but is not limited to, an infection, an autoimmune disease, enzymatic deficiencies (including primary (congenital) enzymatic deficiency and enzymatic deficiencies secondary to functional gut disorders), diabetes, obesity, metabolic disorders, intestinal bacterial overgrowth, enteric infection, bacterial vaginosis, short bowel syndrome, inflammatory bowel disease, irritable bowel syndrome, small bowel syndrome, Celiac disease, gluten intolerance, colitis, peptic ulcer, gastritis, polyps, hemorrhoids, or cirrhosis.

[0093] The various compositions disclosed herein may comprise one or more drugs, biologics, or active agents, which are used interchangeably herein and refer to a chemical substance or compound that induces a desired pharmacological or physiological effect, and includes agents that are therapeutically effective, prophylactically effective, or cosmetically effective. “Drug,” “biologic,” and “active agent” include any pharmaceutically acceptable, pharmacologically active derivatives and analogs of those drugs, biologics, and active agents specifically mentioned herein, 15 IPTS / 200107384.1Attorney Matter No. TNZ-017WO including, but not limited to, salts, esters, amides, prodrugs, active metabolites, inclusion complexes, analogs, and the like. Suitable drugs, biologics, and active agents may include, but are not limited to, alcohol deterrents; amino acids; ammonia detoxicants; anabolic agents; analeptic agents; analgesic agents; androgenic agents; anesthetic agents; anorectic compounds; anorexic agents; antagonists; anti-allergic agents; anti-amebic agents; anti-anemic agents; anti-anginal agents; anti-anxiety agents; anti-arthritic agents; anti-atherosclerotic agents; anti-bacterial agents; anti-cancer agents, including antineoplastic drugs, and anti-cancer supplementary potentiating agents; anticholinergics; anticholelithogenic agents; anti-coagulants; anti-coccidal agents; anti- convulsants; anti-depressants; anti-diabetic agents; anti-diarrheals; anti-diuretics; antidotes; anti- dyskinetics agents; anti-emetic agents; anti-epileptic agents; anti-estrogen agents; anti-fibrinolytic agents; anti-fungal agents; anti-glaucoma agents; anti-hemophilic agents; anti-hemorrhagic agents; antihistamines; anti-hyperlipidemic agents; anti-hyperlipoproteinemic agents; antihypertensive agents; anti-hypotensives; anti-infective agents such as antibiotics and antiviral agents; anti- inflammatory agents, both steroidal and non-steroidal; anti-keratinizing agents; anti-malarial agents; antimicrobial agents; anti-migraine agents; anti-mitotic agents; anti-mycotic agents; antinauseants; antineoplastic agents; anti-neutropenic agents; anti-obsessional agents; anti- parasitic agents; antiparkinsonism drugs; anti-pneumocystic agents; anti-proliferative agents; anti- prostatic hypertrophy drugs; anti-protozoal agents; antipruritics; anti-psoriatic agents; antipsychotics; antipyretics; antispasmodics; anti-rheumatic agents; anti-schistosomal agents; anti- seborrheic agents; anti-spasmodic agents; anti-thrombotic agents; anti-tubercular agents; antitussive agents; anti-ulcerative agents; anti-urolithic agents; antiviral agents; GERD medications, anxiolytics; appetite suppressants; attention deficit disorder (ADD) and attention deficit hyperactivity disorder (ADHD) drugs; bacteriostatic and bactericidal agents; benign prostatic hyperplasia therapy agents; blood glucose regulators; bone resorption inhibitors; bronchodilators; carbonic anhydrase inhibitors; cardiovascular preparations including anti-anginal agents, anti-arrhythmic agents, beta-blockers, calcium channel blockers, cardiac depressants, cardiovascular agents, cardioprotectants, and cardiotonic agents; central nervous system (CNS) agents; central nervous system stimulants; choleretic agents; cholinergic agents; cholinergic agonists; cholinesterase deactivators; coccidiostat agents; cognition adjuvants and cognition enhancers; cough and cold preparations, including decongestants; depressants; diagnostic aids; diuretics; dopaminergic agents; ectoparasiticides; emetic agents; enzymes which inhibit the formation of plaque, calculus or dental caries; enzyme inhibitors; estrogens; fibrinolytic agents; fluoride anticavity / antidecay agents; free oxygen radical scavengers; gastrointestinal motility agents; genetic materials; glucocorticoids; gonad-stimulating principles; hemostatic agents; herbal remedies; histamine H2 receptor antagonists; hormones; hormonolytics; hypnotics; 16 IPTS / 200107384.1Attorney Matter No. TNZ-017WO hypocholesterolemic agents; hypoglycemic agents; hypolipidemic agents; hypotensive agents; immunizing agents; immunomodulators; immunoregulators; immunostimulants; immunosuppressants; impotence therapy adjuncts; inhibitors; keratolytic agents; leukotriene inhibitors; liver disorder treatments; metal chelators such as ethylenediaminetetraacetic acid, tetrasodium salt; mitotic inhibitors; mood regulators; mucolytics; mucosal protective agents; muscle relaxants; mydriatic agents; narcotic antagonists; neuroleptic agents; neuromuscular blocking agents; neuroprotective agents; nicotine; NMDA antagonists; non-hormonal sterol derivatives; nutritional agents, such as vitamins, essential amino acids and fatty acids; ophthalmic drugs such as antiglaucoma agents; oxytocic agents; pain relieving agents; parasympatholytics; peptide drugs; plasminogen activators; platelet activating factor antagonists; platelet aggregation inhibitors; post-stroke and post-head trauma treatments; potentiators; progestins; prostaglandins; prostate growth inhibitors; proteolytic enzymes as wound cleansing agents; prothyrotropin agents; psychostimulants; psychotropic agents; radioactive agents; regulators; relaxants; repartitioning agents; scabicides; sclerosing agents; sedatives; sedative-hypnotic agents; selective adenosine A1 antagonists; serotonin antagonists; serotonin inhibitors; serotonin receptor antagonists; steroids, including progestogens, estrogens, corticosteroids, androgens and anabolic agents; smoking cessation agents; stimulants; suppressants; sympathomimetics; synergists; thyroid hormones; thyroid inhibitors; thyromimetic agents; tranquilizers; tooth desensitizing agents; tooth whitening agents such as peroxides, metal chlorites, perborates, percarbonates, peroxyacids, and combinations thereof; unstable angina agents; uricosuric agents; vasoconstrictors; vasodilators including general coronary, peripheral and cerebral; vulnerary agents; wound healing agents; xanthine oxidase inhibitors; and the like.

[0094] Antibiotic refers to a chemical substance capable of treating bacterial infections by inhibiting the growth of, or by destroying existing colonies of bacteria and other microorganisms.

[0095] Anti-inflammatory refers to an active agent that reduces inflammation and swelling.

[0096] Chemotherapeutic agent refers to a chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer. In one example, a chemotherapeutic agent is a radioactive compound. In one example, a chemotherapeutic agent is a biologic, such as a monoclonal antibody. Chemotherapy refers to use of a chemotherapeutic agent.

[0097] Radiation therapy refers to use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether.

[0098] The various compositions disclosed herein may comprise an effective amount of a drug, biologic, or active agent. Effective amount refers to an amount of a drug, biologic, or active agent (alone or with one or more other active agents) sufficient to induce a desired response, such as to 17 IPTS / 200107384.1Attorney Matter No. TNZ-017WO prevent, treat, reduce and / or ameliorate a condition. An effective amount of an active agent, alone or with one or more other active agents, can be determined in many different ways, such as assaying for a reduction in of one or more signs or symptoms associated with the condition in the subject or measuring the level of one or more molecules associated with the condition to be treated.

[0099] The various compositions disclosed herein may comprise various pharmaceutically acceptable excipients. As used herein, a pH adjuster or modifier refers to a compound or buffer used to achieve desired pH control in a formulation. Exemplary pH modifiers include acids (e.g., acetic acid, adipic acid, carbonic acid, citric acid, fumaric acid, phosphoric acid, sorbic acid, succinic acid, tartaric acid), bases (e.g., magnesium oxide, tribasic potassium phosphate), and pharmaceutically acceptable salts thereof.

[0100] Pharmaceutically acceptable carriers useful in this disclosure are those conventionally known in the art. The nature of the carrier can depend on the particular mode of administration being employed. For instance, oral applications usually include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like, as a vehicle. In addition to biologically-neutral carriers, oral compositions may also contain auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, and the like.

[0101] Antioxidant refers to a compound that inhibits oxidation or reactions promoted by oxygen or peroxides.

[0102] Mucoadhesive refers to a substance that strongly attaches to mucosa upon hydration without any additional adhesive material, and remains adhered to the tissue in vivo.

[0103] Safe Harbor Sites

[0104] In some embodiments, the genome of the engineered yeast cell contains safer harbor sites. As used herein, safe harbor sites are intragenic or extragenic regions of the genome that are able to accommodate the expression of integrated heterologous nucleic acid without adverse effects on the host cell. In some embodiments, safe harbor sites are amenable for genetic manipulation, afford advantages such as biocontainment, genetic stability of integrated alleles, high expression of integrated alleles, and low interference in the transcription of the integrated allege at the safe- harbor site or neighboring genes due to chromatin related positional effects and epigenetic effects. In some embodiments, safe harbor sites include one or more safe harbor sites selected from the group comprising a long terminal repeat (LTR) of a transposable element (Ty element), a non- essential gene, a metabolic auxotrophic gene, and a transcriptionally inactive region in the genome.

[0105] In some embodiments, a non-essential gene is any gene where the impairment or deletion of said gene does not substantially negatively impact the yeast, such as not impacting the growth 18 IPTS / 200107384.1Attorney Matter No. TNZ-017WO of the yeast. In some embodiments, a non-essential gene may include [to align with list of gene added to claims or remove this sentence if not needed].

[0106] In some embodiments, metabolic auxotrophic genes are selected from the group comprising LEU2 (YCL018W), HIS3 (YOR202W), and URA3 (YEL021W). The disruption of these genes can result in one or more metabolic auxotrophies in the engineered yeast strain and limit its growth to environments that have an exogenous supply of leucine, histidine, and / or uracil. This prevents the engineered strain from surviving for long periods in environments where these metabolites are not available, potentially rendering it bio-contained.

[0107] In some embodiments, the safe harbor sites include one or more safe harbor sites from a long terminal repeat (LTR) of a Ty element and a metabolic auxotrophic gene. In some embodiments, long terminal repeat (LTR) of transposable (Ty) elements are sites for integration of recombinant alleles for production of heterologous proteins. These loci are amenable to genetic manipulation, show high expression, low epigenetic interference, and are genetically stable. There are more than 150 such sites in the haploid S. boulardii genome (Khatri et al. 2017) that can be targeted for integration of recombinant alleles encoding production of payload proteins.

[0108] Since, S. boulardii is a naturally diploid organism, each target site exists as two copies and potentially over 300 copies of a payload protein can be genomically integrated into the S. boulardii genome.

[0109] In some embodiments, safe harbor sites have a 5’ integration point and a 3’ integration point. In some embodiments, for the S. boulardii genome, exemplary 5’ integration points and 3’ integration points are provided in Table 1. Table 1.5’ integration points and 3’ integration points Gene at integration h i i i s s s19 IPTS / 200107384.1Attorney Matter No. TNZ-017WO Gene at integration Chr. Sequence 5’ of insertion point Sequence 3’ of insertion point site (S. cerevisiae N t m ti Function Ty nt Ty nt Ty nt Ty nt Ty nt Ty nt Ty nt Ty nt Ty nt Ty nt Ty nt20 IPTS / 200107384.1Attorney Matter No. TNZ-017WO Gene at integration Chr. site (S. cerevisiae N Sequence 5’ of insertion point Sequence 3’ of insertion point t m ti Function Ty nt ble.arbor sites. In some embodiments, the integration is targeted integration. In the context of the present disclosure, a targeted integration refers to integration at a predetermined loci. In contrast, non- targeted integration may refer to, for example, integration at a random loci (such as with transient transfection procedures) or at a randomly selected loci (i.e., a loci that was not subject to analysis prior to selection). In some embodiments, analysis of a loci includes embodiments discloses herein, such as analysis of the distance from centromeres and / or telomeres, determining if neighboring genes are essential genes, and / or analyzing chosen sequences for potential off-target integrations. In some embodiments, the heterologous nucleic acid is integrated at about 1 or more, about 2 or more, about 3 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, about 100 or more, about 105 or more, about 110 or more, about 115 or more, about 120 or more, about 125 or more, about 130 or more, about 135 or more, about 140 or more, about 145 or more, or about 150 or more safe harbor sites. In some embodiments, the heterologous nucleic acid is integrated at about 3 or more, about 5 or more, about 10 or more, about 15 or more, or about 20 or more safe harbor sites. In some embodiments, the heterologous nucleic acid is integrated at about 10 or more or about 15 or more safe harbor sites. In some embodiments, the heterologous nucleic acid is integrated at about 14 safe harbor sites. In some embodiments, the heterologous nucleic acid is integrated at 14 safe harbor sites.

[0111] In some embodiments, a diploid yeast strain is used. In some embodiments, integration in a diploid yeast cell results in twice the copy number of the heterologous nucleic acid. In some embodiments, integration in a diploid yeast cell results in about 2 or more, about 4 or more, about 6 or more, about 10 or more, about 14 or more, about 18 or more, about 20 or more, about 22 or more, about 24 or more, about 26 or more, about 28 or more, about 30 or more, about 40 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 110 or more, about 120 or more, about 130 or more, about 140 or more, about 21 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 150 or more, about 160 or more, about 170 or more, about 180 or more, about 190 or more, about 200 or more, about 210 or more, about 220 or more, about 230 or more, about 240 or more, about 250 or more, about 260 or more, about 270 or more, about 280 or more, about 290 or more, or about 300 or more genomically integrated copies of the heterologous nucleic acid. In some embodiments, the heterologous nucleic acid is integrated at about 1 or more, about 2 or more, about 3 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, about 100 or more, about 105 or more, about 110 or more, about 115 or more, about 120 or more, about 125 or more, about 130 or more, about 135 or more, about 140 or more, about 145 or more, or about 150 or more safe harbor sites in a diploid cell, resulting in about 2 or more, about 4 or more, about 6 or more, about 10 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 110 or more, about 120 or more, about 130 or more, about 140 or more, about 150 or more, about 160 or more, about 170 or more, about 180 or more, about 190 or more, about 200 or more, about 210 or more, about 220 or more, about 230 or more, about 240 or more, about 250 or more, about 260 or more, about 270 or more, about 280 or more, about 290 or more, or about 300 or more genomically integrated copies of the heterologous nucleic acid. Heterologous Nucleic Acid

[0112] In some embodiments, a heterologous nucleic acid is genomically integrated into one or more safe harbor sites. In some embodiments, the heterologous nucleic acid comprises a nucleic acid sequence that encodes a payload protein. In some embodiments, the payload protein is an enzyme, a growth factor, insulin, an incretin, a cytokine, an antibody or fragment thereof, a peptide, an antimicrobial peptide, a mucosal protein, an agricultural product, a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, a nutritional protein, an antiviral, an enzyme inhibitor, or a hormone.

[0113] In some embodiments, the enzyme is selected from the group comprising invertase, isomaltase, lactase, lysozyme, xylose-isomerase, fructose, gluco-amylase, alkaline phosphatase, and An-PEP. In some embodiments, the enzyme is invertase. In some embodiments, the heterologous nucleic acid comprises a sequence encoding invertase. In some embodiments, the enzyme is isomaltase. In some embodiments, the heterologous nucleic acid comprises a sequence encoding isomaltase. In some embodiments, the enzyme is lactase. In some embodiments, the heterologous nucleic acid comprises a sequence encoding lactase. In some embodiments, the enzyme is lysozyme. In some embodiments, the heterologous nucleic acid comprises a sequence 22 IPTS / 200107384.1Attorney Matter No. TNZ-017WO encoding lysozyme. In some embodiments, the enzyme is xylose-isomerase. In some embodiments, the heterologous nucleic acid comprises a sequence encoding xylose-isomerase. In some embodiments, the enzyme is fructose. In some embodiments, the heterologous nucleic acid comprises a sequence encoding fructose. In some embodiments, the enzyme is gluco-amylase. In some embodiments, the heterologous nucleic acid comprises a sequence encoding gluco-amylase. In some embodiments, the enzyme is An-PEP. In some embodiments, the heterologous nucleic acid comprises a sequence encoding An-PEP.

[0114] In some embodiments, the incretin is selected from the group comprising GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, and nesfatin.

[0115] In some embodiments the cytokine is selected from the group comprising IL-2, IL-10, IL- 21, IL-22, and TGFβ. In some embodiments the cytokine is a human cytokine or a mouse cytokine. In some embodiments the cytokine is IL-2. In some embodiments the cytokine is IL-10. In some embodiments the cytokine is IL-21. In some embodiments the cytokine is IL-22. In some embodiments the cytokine is TGFβ.

[0116] Table A below lists various amino acids of payloads. Table A SEQ ID NO Amino acid sequence DescriptionIPTS / 200107384.1Attorney Matter No. TNZ-017WO GAWYVWNRTELMQASLDQSVTHLMGLFEPGDTKYEIHRDPTLDPSLME MTEAALRLLSRNPRGFYLFVEGGRIDHGHHEGVAYQALTEAVMFDDAIE RAGQLTSEEDTLTLVTADHSHVFSFGGYTLRGSSIFGLAPSKAQDSKAYT r24 IPTS / 200107384.1Attorney Matter No. TNZ-017WO KAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIR NHHHHHH 86 SPG GT SENSCTHFPGNLPNMLRDLRDAFSRVKTFF MKD25 IPTS / 200107384.1Attorney Matter No. TNZ-017WO APAPASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTF FQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEV MPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENK26 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 97 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKD Il-10 dimer QLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAEN variant 17 QDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQ27 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 103 SPGQGTQSENSCTHFPGYLPNMLRDLRDAFSRVKTFFQMKD Il-10 dimer QLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAEN variant 22 QDPDIKAHVQSLGENLKDLRLWLRRCHRFLPCENKSKAVE

[0117] In some embodiments, the cytokine is IL-10. In some embodiments, the IL-10 payload comprises a dimer of IL-10. In some embodiments, an IL-10 dimer comprises SEQ ID NO: 71. In some embodiments, the IL-10 dimer comprises any one of SEQ ID NOs: 71 or 81-103. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 81. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 82. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 83. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 84. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 85. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 86. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 87. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 88. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 89. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 90. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 91. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 92. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 93. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 94. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 95. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 28 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 96. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 97. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 98. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 99. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 100. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 101. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 102. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 103. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 104. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 105. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 106. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 107. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 108.

[0118] In some embodiments, the cytokine is IL-21. In some embodiments, IL-21 comprises SEQ ID NO: 72. In some embodiments, the cytokine is IL-22. In some embodiments, IL-22 comprises SEQ ID NO: 73. In some embodiments, IL-22 comprises SEQ ID NO: 74. In some embodiments, IL-22 comprises SEQ ID NO: 104.

[0119] In some embodiments, the cytokine is IL-2. In some embodiments, IL-2 comprises any one of SEQ ID NOs: 105-108. In some embodiments, IL-2 comprises SEQ ID NO: 105. In some embodiments, IL-2 comprises SEQ ID NO: 106. In some embodiments, IL-2 comprises SEQ ID NO: 107. In some embodiments, IL-2 comprises SEQ ID NO: 108.

[0120] In some embodiments, the payload is an enzyme. In some embodiments, the enzyme is alkaline phosphatase. In some embodiments, the alkaline phosphatase is human intestinal alkaline phosphatase (hIAP). In some embodiments, the human intestinal alkaline phosphatase (hIAP) comprises SEQ ID NO: 76. In some embodiments, the alkaline phosphatase is yeast alkaline phosphatase (yAP). In some embodiments, the yeast alkaline phosphatase (yAP) comprises SEQ ID NO: 75.

[0121] In some embodiments, the peptide is selected from the group comprising cholecystokinin and Lumenal cholecystokinin release factor (LCRF). In some embodiments, the peptide is cholecystokinin. In some embodiments, the peptide is LCRF.

[0122] In some embodiments, the mucosal protein is selected from the group comprising trefoil factor, Reg3 protein, and superoxide dismutase.

[0123] In some embodiments, the agricultural product is selected from the group comprising pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, and fertilizer.

[0124] It is to be understood that the preceding payload proteins were exemplary only and are not meant to be limiting in any way. For example, payload proteins not recited herein but that have similar activity to those recited herein are also contemplated and fall within the scope of the present disclosure. As a non-limiting example, alkaline phosphatase is provided herein as a non-limiting 29 IPTS / 200107384.1Attorney Matter No. TNZ-017WO example of a payload protein. Thus, alternative protein payloads with similar capacity to neutralize endotoxin activity are also contemplated. Such proteins would include, but not be limited to, human alkaline phosphatase, yeast alkaline phosphatase, bovine alkaline phosphatase, Cobetia amphilecti alkaline phosphatase, Paernibacillus lentus alkaline phosphatase, human lactoferrin (LF-22), human cathelicidin (LL-37), human β-defensin-3 (HβD-3), human α-defensin 5 (HD5), human acyloxyacyl hydrolase (AOAH), Limulus polyphemus anti-lipopolysaccharide factor (ALF-L), tachypleus plasma lectin 2 (TPL2), human bactericidal permeability-increasing protein (BPI), and Crassostrea gigas bactericidal permeability-increasing protein (CgBPI).

[0125] In some embodiments, the heterologous nucleic acid further comprises a nucleic acid sequence that encodes for a synthetic signal peptide. In some embodiments, the synthetic signal peptide is a pre-protein signal peptide, a pro-protein signal peptide, or a combination thereof as provided for herein.

[0126] In some embodiments, the heterologous nucleic acid comprises a homology arm at the 5’ end and a homology arm at the 3’ end of the heterologous nucleic acid. In some embodiments, the homology arms are designed depending on the method of genome integration. In some embodiments, the homology arms can each vary in length between 10 nucleotides and 2,000 nucleotides. In some embodiments, the homology arms can each vary in length between 30 nucleotides and 200 nucleotides.

[0127] In some embodiments, the homology arm at the 5’ end and the homology arm at the 3’ end of the heterologous nucleic acid comprise a sequence complementary to the 5’ insertion site and 3’ insertion stie, respectively.

[0128] In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-3 and 7-18. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85% sequence identity to any one of SEQ ID NOs: 1-3 and 7-18. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1-3 and 7-18. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 95% sequence identity to any one of SEQ ID NOs: 1-3 and 7-18. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 98% sequence identity to any one of SEQ ID NOs: 1-3 and 7-18. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 99% sequence identity to any one of SEQ ID NOs: 1-3 and 7-18. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence according to any one of SEQ ID NOs: 1-3 and 7-18. In some 30 IPTS / 200107384.1Attorney Matter No. TNZ-017WO embodiments, the homology arm at the 5’ end consists of a complementary sequence according to any one of SEQ ID NOs: 1-3 and 7-18.

[0129] In some embodiments, wherein the homology arm at the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4-6 and 19-30. In some embodiments, wherein the homology arm at the 3’ end comprises a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 4-6 and 19-30. In some embodiments, wherein the homology arm at the 3’ end comprises a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 4-6 and 19-30. In some embodiments, wherein the homology arm at the 3’ end comprises a sequence with at least 95% sequence identity to any one of SEQ ID NOs: 4-6 and 19-30. In some embodiments, wherein the homology arm at the 3’ end comprises a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 4-6 and 19-30. In some embodiments, wherein the homology arm at the 3’ end comprises a sequence with at least 99% sequence identity to any one of SEQ ID NOs: 4-6 and 19-30. In some embodiments, wherein the homology arm at the 3’ end comprises a sequence of any one of SEQ ID NOs: 4-6 and 19-30. In some embodiments, wherein the homology arm at the 3’ end consists of a sequence of any one of SEQ ID NOs: 4-6 and 19-30.

[0130] In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 1 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 4. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 2 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 5. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 3 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 6. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 7 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 19. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm 31 IPTS / 200107384.1Attorney Matter No. TNZ-017WO at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 8 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 20. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 9 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 21. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 10 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 22. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 11 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 23. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 12 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 24. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 13 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 25. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 14 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 26. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 15 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 27. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the 32 IPTS / 200107384.1Attorney Matter No. TNZ-017WO homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 16 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 28. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 17 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 29. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise a pair of sequences identified in Table 1. In some embodiments, the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity SEQ ID NO: 18 and the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 30.

[0131] In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into one or more safe harbor sites with heterologous nucleic acids integrated at one or more of the integration sites identified in Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 2 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 3 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 4 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 5 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 6 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 7 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 8 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 9 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 10 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 11 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 12 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 13 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell comprises a heterologous nucleic acid genomically integrated into 14 safe harbor sites of Table 1. In some embodiments, an engineered yeast cell 33 IPTS / 200107384.1Attorney Matter No. TNZ-017WO comprises a heterologous nucleic acid genomically integrated into 15 safe harbor sites of Table 1.

[0132] In some embodiments, the heterologous nucleic acid comprises a constitutive or inducible promoter. In some embodiments, a constitutive promoter may be PALD6, PCYC1, PHXT7, PSSA1, PTDH3, PTEF1, or PTPI1. In some embodiments, the constitutive or inducible promoter is a TEF1 promoter.the heterologous nucleic acid comprises one or two terminator sequences. In some embodiments, the one or two terminator sequences are each a CYC1 terminator or a ADH1 terminator. In some embodiments, the heterologous nucleic acid comprises a terminator sequence 5’ to the sequence encoding a payload protein. In some embodiments, the heterologous nucleic acid comprises a terminator sequence 3’ to the sequence encoding a payload protein. In some embodiments, the heterologous nucleic acid comprises a terminator sequence 5’ to the sequence encoding a payload protein and a terminator sequence 3’ to the sequence encoding a payload protein.

[0134] In some embodiments, the heterologous nucleic acid comprises a nucleic acid sequence that encodes invertase and is integrated at 14 safe harbor sites.

[0135] In some embodiments, the heterologous nucleic acid comprises the following elements operably linked from 5’ to 3’: a CYC1 terminator sequence, a TEF1 promoter sequence, a Sb propeptide sequence, a sequence that encodes invertase, and a ADH1 terminator sequence. Genome Editing Systems

[0136] In some embodiments, provided herein, to genomically integrate a heterologous nucleic acid at one or more harbor sites, a nuclease capable of generating a break in the 5’ integration site or 3’ integration site of each of the one or more harbor sites is utilized.

[0137] In some embodiments, the nuclease is a nuclease selected from the group comprising TALEN, ZFN, a Cas protein, and HO endonuclease.

[0138] In some embodiments, the nuclease is a Cas protein. In some embodiments, the Cas protein is used as part of a system comprising a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) and / or a trans-activating crRNA (tracrRNA), wherein the crRNA contains a target-specific nucleotide region complementary to a region of a strand of the target (i.e., integration site) double-stranded nucleic acid. In some embodiments, the system is selected from the group consisting of a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, and a Type III CRISPR-Cas system. In some embodiments, the Cas protein is used with one or more guideRNAs (gRNAs). In some embodiments, the Cas protein is used with two or more guideRNAs (gRNAs). In some embodiments, the two or more gRNAs target one or more integration sites. 34 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0139] In some embodiments, the nuclease is Cas3, Cas9, Cas12 protein or a variant thereof. In some embodiments, the nuclease is Cas9. Synthetic Signal Peptides

[0140] In some embodiments, the heterologous nucleic acid comprises a sequence encoding synthetic signal peptide that increases secretion of a payload. In some embodiments, the synthetic signal peptide comprises one or more of a synthetic pre-protein signal peptide and pro-protein signal peptide. In any embodiment, a native pre- or pro-protein signal peptide may be combined with a synthetic signal peptide, provided at least one of the pre- and pro-protein signal peptide is synthetic. In some embodiments, recombinant polypeptides are provided comprising a synthetic signal peptide and a payload protein, wherein the synthetic signal peptide is fused, either directly or indirectly, to the payload protein. In some embodiments, the synthetic signal peptide is fused directly to the protein of interest. In some embodiments, heterologous nucleic acid comprises a sequence encoding a synthetic pre-protein signal peptide and a payload protein. For example, FIG. 1 depicts a construct that represents a heterologous nucleic acid with a sequence element encoding a synthetic signal peptide 5’ to the sequence encoding a payload protein..

[0141] Table 2 below lists various amino acid sequences of pre-protein signal peptides and pro- protein signal peptides. In some embodiments, any pre-protein signal peptide or pro-protein signal peptide provided in Table 2 can be used in the heterologous nucleic acid. Table 2 Pre- SEQ ID Amino Acid Se uence and / or ro ro ro ro ro35 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0142] Any synthetic pre-protein signal peptide may be combined with any pro-protein signal peptide. In some embodiments, the signal peptide comprises: MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), or MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36).

[0143] In some embodiments, the signal peptide comprises MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), or MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36).

[0144] In some embodiments, the signal peptide comprises MKFKLTLLAAILAYANTVLVAS (SEQ ID NO: 62) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTLLAAILAYANTVLVAS (SEQ ID NO: 62) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), or MKFKLTLLAAILAYANTVLVAS (SEQ ID NO: 62) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36).

[0145] In some embodiments, the signal peptide comprises MKFKLTLLAALSALAALALAAP (SEQ ID NO: 63) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTLLAALSALAALALAAP (SEQ ID NO: 63) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), or 36 IPTS / 200107384.1Attorney Matter No. TNZ-017WO MKFKLTLLAALSALAALALAAP (SEQ ID NO: 63) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36). In some embodiments, the signal peptide comprises MKFKLTIFAALLALAALALAAP (SEQ ID NO: 64) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTIFAALLALAALALAAP (SEQ ID NO: 64) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), or MKFKLTIFAALLALAALALAAP (SEQ ID NO: 64) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36).

[0146] In some embodiments, the signal peptide comprises the sequence of MKFKLTLLAALLALAALVLAASEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASR HKRDV (SEQ ID NO: 37). In some embodiments, the signal peptide comprises the sequence of MKFKSALFAALLALAALVLAAPEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASR HKRDV (SEQ ID NO: 65). In some embodiments, the signal peptide comprises the sequence of MKFKLTLLAAILAYANTVLVASEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRH KRDV (SEQ ID NO: 66). In some embodiments, the signal peptide comprises the sequence of MKFKLTLLAALSALAALALAAPEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASR HKRDV (SEQ ID NO: 67). In some embodiments, the signal peptide comprises the sequence of MKFKLTIFAALLALAALALAAPEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRH KRDV (SEQ ID NO: 68).

[0147] Any synthetic pre-protein or pro-protein signal peptide may be combined with some or all of a known signal peptide. Examples of known signal peptides that may be combined with any of SEQ ID NOs: 31-36 and 61-64 in Table 2 to generate a synthetic signal peptide include, but are not limited to, HSp150, PHO5, SUC2, KILM1, GGP1, SUN, PLB, CRH, EXG, AGA2, HAS pre- pro, PIR1, XPR2 pre, XPR2 pre-pro, pGKL, SCW, and DSE. Other non-limiting examples of pre- protein signal peptides, pro-protein signal peptides, and combinations thereof that are within the scope of the present disclosure include those described in International Patent Publication Serial No. WO2022 / 192675 and U.S. Patent Publication Serial No. US2024 / 0174722, each of which are incorporated by reference herein in their entirety.

[0148] One who is skilled in the art will be able to develop a nucleic acid that encodes for the expression of any one of SEQ ID NOs.31-36 and 61-64 or fusions thereof. Any such nucleic acid is within the scope of the present disclosure.

[0149] The synthetic signal peptides disclosed herein are optimized for use in yeast and can be used to induce expression of any protein. Particular examples of suitable yeast species are provided herein below. As noted above, Table 2 discloses amino acid sequences, however, in any aspect and embodiment, any of the sequences in Table 2 may be modified with conservative amino acid 37 IPTS / 200107384.1Attorney Matter No. TNZ-017WO substitutions to produce active variants that maintain the characteristics and functionality of the primary sequence. These conservative amino acid substitutions can be generally described by the Formulas below, which encapsulate the consensus sequence as well as the variant sequences. The various Formulas detailing the variant sequences will now be described. In some embodiments, a pre-protein signal peptide is provided.. Synthetic Pre-Protein Signal Peptides and Their Use in Saccharomyces Yeast

[0150] In some embodiments, S. cerevisiae or S. boulardii yeast may be genetically modified with a nucleic acid encoding the expression of a payload protein comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO. 31, 32, 33, 61, 62, 63, or 64. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein. In some embodiments, any nucleic acid encoding for SEQ ID NO. 31, 32, 33, 61, 62, 63, or 64 may be utilized to induce expression of the synthetic pre-protein signal peptide. One of skill in the art will understand how to develop a suitable nucleic acid that will induce expression of a synthetic signal peptide comprising an amino acid sequence of SEQ ID NO.31, 32, 33, 61, 62, 63, or 64. In some embodiments, a pre-protein signal peptide comprising an amino acid sequence of SEQ ID NO.31, 32, 33, 61, 62, 63, or 64 may be fused directly or indirectly to a native constitutive pro-protein signal peptide. In some embodiments, a pre-protein signal peptide comprising an amino acid sequence of SEQ ID NO. 31, 32, 33, 61, 62, 63, or 64 may be fused directly or indirectly to a synthetic signal peptide as disclosed herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the native or synthetic pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the native or synthetic pro-protein signal peptide via, for example, a peptide linker as provided for herein.

[0151] In some embodiments, a recombinant polypeptide comprising a synthetic pre-protein signal peptide comprising an amino acid sequence of SEQ ID NO. 31, 32, 33, 61, 62, 63, or 64 and a payload protein is provided. In some embodiments, inclusion of the synthetic pre-protein signal peptide comprising an amino acid sequence of SEQ ID NO. 31, 32, 33, 61, 62, 63, or 64 will result in the payload protein being more readily secreted by the yeast in which it is produced. Accordingly, in another embodiment, a method of producing a payload protein with Saccharomyces yeast is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide comprising an amino acid sequence of SEQ ID NO. 31, 32, 33, 61, 62, 63, or 64; genetically modifying the Saccharomyces yeast with the nucleic acid, thereby generating 38 IPTS / 200107384.1Attorney Matter No. TNZ-017WO engineered yeast; and culturing the engineered yeast under effective conditions to express the recombinant polypeptide. In some embodiments, the nucleic acid molecule encoding for the amino acid sequence of SEQ ID NO.31, 32, 33, 61, 62, 63, or 64 is any nucleic acid molecule encoding for said amino acid sequence.

[0152] In some embodiments, an engineered Saccharomyces yeast (e.g., S. boulardii or S. cerevisiae) is provided, wherein the yeast is genetically modified with a nucleic acid molecule encoding the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre- protein signal peptide comprises an amino acid sequence of SEQ ID NO.31, 32, 33, 61, 62, 63, or 64. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro- protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide as provided for herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein. In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an enzyme (e.g., invertase, isomaltase, lactase, alkaline phosphatase, lysozyme, alkaline phosphatase, An-PEP), a growth factor (e.g., IGF-1), insulin, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), a cytokine (e.g., IL-2, IL-10, IL-21, IL-22), an antibody, an antimicrobial peptide), a mucosal protein (e.g., trefoil factor, Reg3 protein, superoxide dismutase), an agricultural product (e.g., pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein. The examples listed are provided for clarity only and are not meant to be limiting in any way. Thus, for example, the current disclosure is not limited to IGF-1 for “growth factor”, but rather encompasses and includes all growth factors known in the art. Methods of Generating Engineered Yeast

[0153] Various suitable signal peptides are disclosed above with specific examples of signal peptides comprising various synthetic pre- and synthetic pro-protein signal detailed in Table 3 below. Table 3 Pre-Protein Pro-ProteinIPTS / 200107384.1Attorney Matter No. TNZ-017WO 32 34 32 35 2

[0154] Other ety or in part into a synthetic sigg p p , p , , 5, SUC2, KILM1, GGP1, SUN, PLB, CRH, EXG, AGA2, HAS pre-pro, PIR1, XPR2 pre, XPR2 pre-pro, pGKL, SCW, and DSE.

[0155] In some embodiments, a method of generating an engineered yeast that expresses a recombinant polypeptide comprising a synthetic signal peptide is provided, the method comprising providing a yeast, contacting the yeast with a nucleic acid molecule encoding the recombinant polypeptide comprising the synthetic signal peptide, and culturing the yeast under conditions suitable to genetically modify the yeast to induce expression of the recombinant polypeptide, thereby creating an engineered yeast.

[0156] The yeast may be any strain of yeast, such as, but not limited to, Saccharomyces (e.g., S. cerevisiae, S. boulardii). In some embodiments, inducing expression of the recombinant polypeptide may be carried out via any expression system known to those skilled in the art. For example, in some embodiments, the method of generating an engineered yeast may comprise preparing a vector containing a nucleic acid (e.g., RNA, DNA) encoding the recombinant polypeptide, transporting the vector to the host yeast (“genetically modifying”), and culturing the yeast under effective conditions to express the recombinant polypeptide. As used herein, the term “vector” refers to a nucleotide molecule capable of transporting other nucleotides to which it has been linked. One exemplary type of vector is a “plasmid”, which represents a circular double stranded DNA loop into which additional DNA sections can be ligated. Another type of vector is a viral vector; wherein additional DNA sections can be ligated with the viral genome. Methods of introducing a DNA into yeast are known to those skilled in the art and may include a 40 IPTS / 200107384.1Attorney Matter No. TNZ-017WO transformation method, a transfection method, an electroporation method, a nuclear injection method, or a carrier such as a liposome, micelle, skin cell, or a fusion method using protoplasts. A recombinant nucleic acid encoding the recombinant polypeptide may be obtained from any source using conventional techniques known to those skilled in the art, including isolation from genomic or cDNA libraries, amplification by PCR, or chemical synthesis.

[0157] In some embodiments, an engineered yeast may be cultured to induce growth of the yeast for a period of time in an environment effective to maintain the health of the yeast, thereby generating a desired amount of recombinant polypeptide comprising the synthetic signal peptide and payload protein. The culturing of yeast is common practice and well known in the art. In general, yeast can be grown in broth or agar in the presence of culture medium comprising bacteriological peptone, yeast extract, and glucose. Supplemental components such as amino acids, buffers, polysaccharides, and salts are sometimes used as well, depending on the strain and application. Engineered yeast may be grown at room temperature or, more effectively, at a temperature of up to about 30℃ to 37℃. Temperature may be used to control the growth of the yeast cells and to regulate the production of the desired recombinant polypeptide. Thus, in some embodiments, the yeast may be grown at a temperature from about 4°C to about 50°C. The recited temperature range includes any temperature range within said range. Thus, in some embodiments, the yeast may be grown at a temperature from about 4°C to about 40°C, from about 10°C to about 50°C, from about 10°C, to about 45°C, from about 15°C, to about 45°C, from about 20°C to about 45°C, from about 25°C to about 45°C, from about 30°C to about 50°C, from about 35°C to about 50°C, from about 37°C to about 50°C, from about 40°C to about 50°C, or from about 45°C to about 50°C. Similarly, the recited ranges include each and every individual temperature within said range. Thus, in some embodiments, the yeast may be grown at a temperature of about 4°C. In some embodiments, the yeast may be grown at a temperature of about 50°C. In some embodiments, the yeast may be grown at a temperature of about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C. Further, those skilled in the art will recognize that further modifications to the growth conditions may be necessary depending on the strain of yeast utilized and the recombinant polypeptide being produced. Such modifications are within the scope of the present application. In any case, secretion of a payload protein by the host yeast will result in its accumulation in the surrounding culture medium, where it may then be 41 IPTS / 200107384.1Attorney Matter No. TNZ-017WO collected, isolated, and / or quantified. Through various intracellular mechanisms, the payload protein will be extracellularly secreted with or without some or all of the synthetic signal peptide to which it was fused.

[0158] In some embodiments, the proteins that may be produced by the engineered yeast include any protein. In some embodiments, the proteins that may be produced by the engineered yeast disclosed herein include, but are not limited to, maltose binding protein (MBP), trefoil factor, mucin, DNase, clotting or blood volumizing factors, insulin and insulin analogs, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), EGFP, PDGF, HB-EGF, α1-antitrypsin, serum albumin, collagen, pepsinogen, tumor necrosis factor, streptokinase, glucagon, lepirudin, desirudin, hirudin, encallantide, IFN-α 2b, antigens, antibodies and antibody fragments thereof (e.g., anti-IL-6R Ab, anti-RSV ab, tetanus toxin fragment C, An-PEP, HIV-1 gp120 (intracellular), HIV-1 gp120 (secret), Bm86 tick gut glytoprotein, murine single-chain antibody, anti-TNF Ab, cancer antibodies, sHBsAg), enzymes (e.g., lysozyme, invertase, galactanase, isomaltase, lactase, chitiniase, xylanase, catalase, D-alanine carboxypeptidase, α-amylase, aspartic proteinase II, galactosidase, horseradish peroxidase, rasburicase, ocriplasmin, pancrelipase, alcohol dehydrogenase (I and II), phosphoglyserate kinase, GADPH, alkaline phosphatase, acid phosphatase), enzyme inhibitors (e.g., Kunitz protease inhibitor, tick anticoagulant protein, ghilanten, tPA Kringle type-2 domain), hormones (e.g., HGH, follicle stimulating hormone, human parathyroid hormone), vaccines (e.g., hepatitis vaccine (I), HPV vaccine), food processing products (e.g., brazzein, chymocin, beta-galactosidase), and cytokines (e.g., IL-2, IL-10, IL-21, IL-22).

[0159] In some embodiments, secretion of a payload protein by a yeast is increased by genetically modifying the yeast to express the payload protein as part of a recombinant polypeptide comprising a synthetic signal peptide as disclosed herein. Accordingly, in some embodiments, an engineered yeast may secrete about 10% to about 200% more of a payload protein than a wild-type yeast. In some embodiments, an engineered yeast may express about 10% to about 50% more, about 20% to about 70% more, about 30% to about 90% more, or about 50% to about 200% more of a payload protein than a wild-type yeast. It is to be understood that any individual percentage of increased payload protein secretion is encompassed within the embodiments described herein. Accordingly, in some embodiments, the yeast may secrete about 10% more of a payload protein. In some embodiments, the yeast may secrete about 20% more of a payload protein. In some embodiments, the yeast may secrete about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200% more of a payload protein than a wild-type yeast, or any percentage falling within any of the recited percentages. 42 IPTS / 200107384.1Attorney Matter No. TNZ-017WO Those of skill in the art would recognize that any change in growth condition during routine optimization for expression of a particular recombinant polypeptide of interest may also affect the amount of payload protein secreted by the engineered yeast. Accordingly, in some embodiment, an engineered yeast may secrete at least 10% more of a payload protein. Accordingly, in some embodiments, an engineered yeast may secrete about 10% more, about 100%, about 500% more, about 1000% more, or about 10,000% more of a payload protein compared to a wild-type yeast or a yeast expressing a transiently transfected plasmid. In some embodiments, secretion is measured by measuring the concentration of the payload protein in the culture media in which the yeast was grown. The concentration may be normalized to optical density to account for variations in growth of the yeast. In some embodiments, secretion is measured by any method known to those skilled in the art for measuring payload protein concentration.

[0160] In some embodiments, the payload protein may be isolated from the culture medium in which the engineered yeast is grown using any methods known to those skilled in the art, such as precipitation from the medium, immunoaffinity chromatography, receptor affinity chromatography, or hydrophobic interaction chromatography. In some embodiments, the payload protein may be isolated by conventional chromatographic methods such as affinity chromatography, size-exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like.

[0161] In some embodiments, a recombinant polypeptide may be designed to comprise a specific affinity peptide, tag, label, or chelate residue that is recognized by a specific binding partner or agent which may aid in isolation. In some embodiments, the recombinant polypeptide variants comprising the additional tag, label, or residue may then be cleaved to obtain the payload protein. Anchoring proteins

[0162] The cell wall of Saccharomyces cerevisiae is composed of glucan, mannoproteins, and a small amount of chitin. Mannoproteins in the yeast cell wall are grouped into two classes. The first class is proteins that can be extracted with sodium dodecyl sulfate (SDS). These proteins are considered to be noncovalently entrapped or associated in the cell wall. The other class is cell wall proteins that cannot be extracted with SDS. These proteins are considered to be covalently bound to cell wall glucan and can be solubilized by incubation with β-1,3-glucanase. Among these proteins are structural cell wall proteins, such as Cwp1p, Cwp2p, Tip1p, and Sed1. These proteins are rich in serine and threonine residues and contain signals for the addition of a glycosylphosphatidylinositol (GPI) anchor. This anchor is transferred to cell wall proteins in the endoplasmic reticulum, and GPI-anchored proteins are transported through the Golgi apparatus to the plasma membrane. 43 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0163] Sed1 is a cell wall protein covalently bound to cell wall glucan. Sed1 is a major structural cell wall protein in stationary-phase cells and plays an important role in cell defense mechanisms in the stationary phase.

[0164] Cell wall protein 2 (CWP2) is a cell wall protein produced by Saccharomyces cerevisiae and Saccharomyces pastorianus. CWP2 is covalently bonded to the cell wall and serves as a significant component of the cell wall structure.

[0165] In some embodiments, a heterologous nucleic acid comprises a sequence encoding anchoring protein that coats the yeast cell surface with the payload. In some embodiments, the anchoring protein that comprises one or more of a synthetic anchoring protein. In some embodiments, the heterologous nucleic acid comprises a sequence encoding synthetic signal peptide that increases secretion of a payload and an anchoring protein. In some embodiment, a native pre- or pro-protein signal peptide may be combined with a synthetic signal peptide, provided at least one of the pre- and pro-protein signal peptide is synthetic, and an anchoring protein.

[0166] In some embodiments, recombinant polypeptides are provided comprising a synthetic signal peptide, an anchoring protein, and a payload protein, wherein the synthetic signal peptide is fused, either directly or indirectly, to the payload protein, and the payload protein is fused, either directly or indirectly, to the anchoring protein. In some embodiments, recombinant polypeptides are provided comprising a synthetic signal peptide, an anchoring protein, and a payload protein, wherein the synthetic signal peptide is fused, either directly or indirectly, to the anchoring protein, and the anchoring protein is fused, either directly or indirectly, to the payload protein. In some embodiments, the synthetic signal peptide is fused directly to the payload protein. In some embodiments, the synthetic signal peptide is fused directly to the anchoring protein. In some embodiments, the payload protein is fused directly to the anchoring protein. In some embodiments, heterologous nucleic acid comprises a sequence encoding a synthetic pre-protein signal peptide, pro-protein signal, a payload protein, and an anchoring protein. For example, FIGs. 11-12 depict construct that represents a heterologous nucleic acid with a sequence element encoding a synthetic signal peptide 5’ to the sequence encoding a payload protein, and a payload protein 5’ to the sequence encoding an anchoring protein.

[0167] Table 4 below lists various amino acid sequences of anchoring proteins. Table 4 SEQ ID NO Amino Acid Sequence DescriptionIPTS / 200107384.1Attorney Matter No. TNZ-017WO 70 ISQITDGQIQATTTATTEATTTAAPSSTVETVSPSSTETISQQTENGAAKAA VGMGAGALAAAAMLL CWP2

[0168] Any synthetic pre-protein signal peptide may be combined with any pro-protein signalme embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 31 and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 31 and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 32 and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 32 and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 33 and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 33 and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 61 and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 61 and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 62 and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 62 and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 63 and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 63 and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 64 and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 64 and an anchoring protein comprising SEQ ID NO: 70.

[0169] In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 31, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 31, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 32, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 32, a pro-protein 45 IPTS / 200107384.1Attorney Matter No. TNZ-017WO sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 33, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 33, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 61, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 61, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 62, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 62, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 63, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 63, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 64, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 64, a pro-protein sequence having SEQ ID NO: 34, and an anchoring protein comprising SEQ ID NO: 70.

[0170] In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 31, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 31, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 32, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 32, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 33, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence 46 IPTS / 200107384.1Attorney Matter No. TNZ-017WO having SEQ ID NO: 33, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 61, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 61, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 62, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 62, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 63, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 63, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 64, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 64, a pro-protein sequence having SEQ ID NO: 35, and an anchoring protein comprising SEQ ID NO: 70.

[0171] In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 31, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 31, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 32, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 32, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 33, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 33, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 61, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid 47 IPTS / 200107384.1Attorney Matter No. TNZ-017WO comprises a pre-protein sequence having SEQ ID NO: 61, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 62, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 62, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 63, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre- protein sequence having SEQ ID NO: 63, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 70. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 64, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 69. In some embodiments, a heterologous nucleic acid comprises a pre-protein sequence having SEQ ID NO: 64, a pro-protein sequence having SEQ ID NO: 36, and an anchoring protein comprising SEQ ID NO: 70.

[0172] The anchoring protein anchors a payload protein to the yeast cell surface. In some embodiments, the payload protein is an enzyme, a growth factor, insulin, an incretin, a cytokine, an antibody or fragment thereof, a peptide, an antimicrobial peptide, a mucosal protein, an agricultural product, a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, a nutritional protein, an antiviral, an enzyme inhibitor, or a hormone.

[0173] In some embodiments, the enzyme is selected from the group comprising invertase, isomaltase, lactase, lysozyme, xylose-isomerase, fructose, gluco-amylase, alkaline phosphatase, and An-PEP. In some embodiments, the enzyme is invertase. In some embodiments, the heterologous nucleic acid comprises a sequence encoding invertase. In some embodiments, the enzyme is isomaltase. In some embodiments, the heterologous nucleic acid comprises a sequence encoding isomaltase. In some embodiments, the enzyme is lactase. In some embodiments, the heterologous nucleic acid comprises a sequence encoding lactase. In some embodiments, the enzyme is lysozyme. In some embodiments, the heterologous nucleic acid comprises a sequence encoding lysozyme. In some embodiments, the enzyme is xylose-isomerase. In some embodiments, the heterologous nucleic acid comprises a sequence encoding xylose-isomerase. In some embodiments, the enzyme is fructose. In some embodiments, the heterologous nucleic acid comprises a sequence encoding fructose. In some embodiments, the enzyme is gluco-amylase. In some embodiments, the heterologous nucleic acid comprises a sequence encoding gluco-amylase. In some embodiments, the enzyme is An-PEP. In some embodiments, the heterologous nucleic acid comprises a sequence encoding An-PEP. 48 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0174] In some embodiments, the incretin is selected from the group comprising GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, and nesfatin.

[0175] In some embodiments the cytokine is selected from the group comprising IL-2, IL-10, IL- 21, IL-22, and TGFβ. In some embodiments the cytokine is IL-2. In some embodiments the cytokine is IL-10. In some embodiments the cytokine is IL-21. In some embodiments the cytokine is IL-22. In some embodiments the cytokine is TGFβ.

[0176] In some embodiments, the cytokine is IL-10. In some embodiments, the IL-10 payload comprises a dimer of IL-10. In some embodiments, an IL-10 dimer comprises SEQ ID NO: 71. In some embodiments, the IL-10 dimer comprises any one of SEQ ID NOs: 71 or 81-103. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 81. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 82. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 83. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 84. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 85. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 86. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 87. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 88. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 89. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 90. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 91. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 92. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 93. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 94. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 95. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 96. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 97. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 98. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 99. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 100. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 101. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 102. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 103. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 104. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 105. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 106. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 107. In some embodiments, the IL-10 dimer comprises SEQ ID NO: 108.

[0177] In some embodiments, the cytokine is IL-21. In some embodiments, IL-21 comprises SEQ ID NO: 72. In some embodiments, the cytokine is IL-22. In some embodiments, IL-22 comprises SEQ ID NO: 73. In some embodiments, IL-22 comprises SEQ ID NO: 74. In some embodiments, IL-22 comprises SEQ ID NO: 104.

[0178] In some embodiments, the cytokine is IL-2. In some embodiments, IL-2 comprises any one of SEQ ID NOs: 105-108. In some embodiments, IL-2 comprises SEQ ID NO: 105. In some 49 IPTS / 200107384.1Attorney Matter No. TNZ-017WO embodiments, IL-2 comprises SEQ ID NO: 106. In some embodiments, IL-2 comprises SEQ ID NO: 107. In some embodiments, IL-2 comprises SEQ ID NO: 108.

[0179] In some embodiments, the payload is an enzyme. In some embodiments, the enzyme is alkaline phosphatase. In some embodiments, the alkaline phosphatase is human intestinal alkaline phosphatase (hIAP). In some embodiments, the human intestinal alkaline phosphatase (hIAP) comprises SEQ ID NO: 76. In some embodiments, the alkaline phosphatase is yeast alkaline phosphatase (yAP). In some embodiments, the yeast alkaline phosphatase (yAP) comprises SEQ ID NO: 75. Methods of their Use

[0180] In some embodiments, an engineered yeast as disclosed herein may be used to deliver one or more of a therapeutic protein, diagnostic protein, or protein-based vaccine to a subject in need thereof. In some embodiments, the engineered yeast as disclosed herein may be used to deliver a payload protein to a specific organ or location within the subject, for example, to a subject’s GI tract, skin, reproductive tract, or the like. In some embodiments the subject may be an animal, such as a companion animal (e.g., dog, cat, rodent, or the like). In some embodiments, the subject may be a livestock animal (e.g., cattle, sheep, horse, pig, goat, or the like). In some embodiments, the subject is a human.

[0181] In some embodiments, an engineered yeast may be used to deliver one or more of a protein- based herbicide, fungicide, bactericide, insecticide, nematicide, miticide, plant growth regulator, plant growth stimulant, or fertilizer in an agricultural environment, such as to crops or plants (such as seeds, roots, corn, tubers, bulbs, slip, rhizome, grass, or vines) or to a plant growth environment (such as topsoil, top dressing, compost, manure, water table, or hydroponic tank).

[0182] In some embodiments, an engineered yeast may be incorporated into a food product, such as bread, dairy, or fermented beverage, to deliver a therapeutic protein, diagnostic protein, protein- based vaccine, an anti-spoilage agent (e.g., bactericide or fungicide), protein-based flavoring agent, protein supplement, or an allergen degrader (e.g., gluten enzyme).

[0183] In some embodiments, an engineered yeast may be used to deliver any protein in any application or environment where fermentation is desired. Further specific uses are described herein below. Therapeutic Compositions and Methods of their Use

[0184] In some embodiments, the engineered yeast may have therapeutic efficacy and as such, may be used to treat a condition, disorder, or disease in a subject. Accordingly, in some embodiments, a method of treating a condition, disorder, or disease in a subject in need thereof in provided, the method comprising administering a composition comprising a therapeutically 50 IPTS / 200107384.1Attorney Matter No. TNZ-017WO effective amount of a protein, wherein the protein is produced in an engineered yeast genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or both of a synthetic pre-protein signal and a synthetic pro-protein signal as disclosed herein. In some embodiments, administering may be performed via any route, such as oral or topical. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered topically.

[0185] In some embodiments, a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic payload protein is provided, wherein the therapeutic payload protein is generated by an engineered yeast genetically with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, as disclosed in any aspect or embodiment herein. In some embodiments, the disease or condition may include, but is not limited to, an infection, an autoimmune disease, enzymatic deficiencies (including primary (congenital) enzymatic deficiency and enzymatic deficiencies secondary to functional gut disorders), diabetes, obesity, metabolic disorders, intestinal bacterial overgrowth, enteric infection, bacterial vaginosis, short bowel syndrome, inflammatory bowel disease, irritable bowel syndrome, small bowel syndrome, Celiac disease, gluten intolerance, colitis, peptic ulcer, gastritis, polyps, hemorrhoids, cirrhosis, endotoxemia, or a cancer. Furthermore, the disease or condition may be caused by, or present with, endotoxemia, such as, but not limited to, aging, anxiety, autism, Alzheimer’s disease, Amyotrophic lateral sclerosis, atherosclerosis, chronic fatigue syndrome, chronic heart disease, cirrhosis, depression, diabetes, exercise-induced enteropathy, HIV enteropathy, metabolic endotoxemia, NSAID enteropathy, nonalcoholic fatty liver disease, inflammatory bowel disease, irritable bowel syndrome, obesity, Parkinson’s disease, sepsis, small intestinal bacterial overgrowth, rheumatoid arthritis, or thrombosis.

[0186] In some embodiments, a composition comprising a therapeutic protein that is produced by any engineered yeast disclosed herein may be formulated for oral, topical, parenteral, or transdermal administration. These compositions may be in form of pill, tablet, capsule, microcapsule, powder, sachet, dragee, gel, liquid, suspension, solution, food product, cream or granule, and may further comprise one or more pharmaceutically acceptable excipients such as, but not limited to, carriers, solvents, co-solvents, emulsifiers, lubricants, disintegrants, binders, fillers, glidants, rheology agents, solubilizers, antimicrobials, antioxidants, preservatives, colorants, flavor agents, emollients, pH modifiers, and the like.

[0187] In some embodiments, food products may include, but are not limited to, a dairy product, a yoghurt, an ice cream, a milk-based drink, a milk-based garnish, a pudding, a milkshake, an ice tea, a fruit juice, a diet drink, a soda, a sports drink, a powdered drink mixture for dietary supplementation, an infant and baby food, a calcium-supplemented orange juice, a sauce or a soup. 51 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0188] In some embodiments, the engineered yeast may be utilized as a conduit for drug delivery to a subject. For example, engineered yeast may be orally administered to a subject to treat a condition, disorder, or disease, wherein the engineered yeast continues to produce and secrete the therapeutic protein within the subject, therefore providing a therapeutic benefit to the subject. Accordingly, in some embodiments, a method of treating a condition, disorder, or disease in a subject in need thereof is provided, the method comprising administering a therapeutically effective amount of engineered yeast as described herein, to the subject. In some embodiments, the therapeutically effective amount of engineered yeast may be orally administered to the subject. In some embodiments, the condition, disorder, or disease may include, but is not limited to, a GI disease or condition, a topical disease or condition, or a mucosal disease or condition. For example, the disease can be a viral (e.g. rotavirus), bacterial, fungal, or parasitic infection (such as, but not limited to intestinal bacterial overgrowth, bacterial vaginosis, an STI), an autoimmune disease (e.g., GBS), an enzymatic or vitamin deficiency (such as lactose intolerance, CSID, Celiac disease / gluten intolerance), a metabolic disorder such as diabetes, an inflammatory GI disease (e.g., irritable bowel syndrome, inflammatory bowel disease, colitis, gastritis, polyps), other GI condition or disease where healing / repair is required (e.g., peptic ulcer), an inflammatory skin condition (e.g. atopic dermatitis, diabetic ulcer), a wound, short bowel syndrome, hemorrhoids, cirrhosis, or a cancer. In some embodiments, administering may be performed via any route, such as oral or topical. The therapeutically effective amount of engineered yeast may be measured in colony forming units (CFUs) and may be any amount, such as from about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, about 104CFUs, about 108CFUs, about 109CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0189] In another embodiment, a pharmaceutical composition comprising an engineered yeast with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, as disclosed in any aspect or embodiment herein, is provided.

[0190] In some embodiments, the composition comprises a Saccharomyces yeast (e.g. S. boulardii or S. cerevisiae) genetically with a heterologous nucleic acid genomically integrated into one or more safe harbor sites. 52 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0191] In some embodiments, the disease or condition is an enzyme deficiency, and the payload protein is an enzyme.

[0192] In some embodiments, the disease or condition is congenital sucrose-isomaltase deficiency and the payload protein is one or both of invertase and isomaltase.

[0193] In some embodiments, the disease or condition is sucrose intolerance secondary to a functional gut disorder and the payload protein is one or both of invertase and isomaltase. In some embodiments, the disease or condition is isomaltase intolerance secondary to a functional gut disorder and the payload protein is one or both of invertase and isomaltase. In some embodiments, the disease or condition is one or both of sucrose and isomaltase intolerance secondary to a functional gut disorder and the payload protein is one or both of invertase and isomaltase.

[0194] In some embodiments, the disease or condition is one or more of gluten intolerance, refractory sprue, or Celiac disease and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubingensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide. In some embodiments, the disease or condition is gluten intolerance and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubingensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide. In some embodiments, the disease or condition is refractory sprue and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubingensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide. In some embodiments, the disease or condition is Celiac disease and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubingensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide.

[0195] In some embodiments, the disease or condition is pancreatitis or exocrine pancreatic insufficiency and the payload protein is selected from one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin. In some embodiments, the disease or condition is pancreatitis and the payload protein is selected from one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin. In some embodiments, the disease or condition is exocrine pancreatic insufficiency and the payload protein is selected from one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin.

[0196] In some embodiments, the disease or condition is enteropeptidase deficiency or enterokinase deficiency and the payload protein is one or all of enteropeptidase, proenteropeptidase, and enterokinase. In some embodiments, the disease or condition is enteropeptidase deficiency and the payload protein is one or all of enteropeptidase, proenteropeptidase, and enterokinase. In some embodiments, the disease or condition is enterokinase deficiency and the payload protein is one or all of enteropeptidase, proenteropeptidase, and enterokinase. 53 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0197] In some embodiments, the disease or condition is small intestinal bacterial overgrowth, inflammatory bowel disease, irritable bowel syndrome, C. difficile infection, cystic fibrosis, necrotizing enterocolitis, and diabetes, and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is small intestinal bacterial overgrowth, and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is inflammatory bowel disease and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is irritable bowel syndrome and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is C. difficile infection and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is cystic fibrosis and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is necrotizing enterocolitis and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is diabetes and the payload protein is intestinal alkaline phosphatase.

[0198] In some embodiments, the disease or condition is short bowel syndrome and the payload protein is IGF-1, GLP-2, or a synthetic derivative of GLP-2. In some embodiments, the disease or condition is short bowel syndrome and the payload protein is IGF-1. In some embodiments, the disease or condition is short bowel syndrome and the payload protein is GLP-2. In some embodiments, the disease or condition is short bowel syndrome and the payload protein is a synthetic derivative of GLP-2.

[0199] In some embodiments, the disease or condition is lactose sensitivity or lactose intolerance and the payload protein is lactase. In some embodiments, the disease or condition is lactose sensitivity and the payload protein is lactase. In some embodiments, the disease or condition is lactose intolerance and the payload protein is lactase.

[0200] In some embodiments, the disease or condition is trehalose sensitivity or lactose intolerance and the payload protein is trehalase.

[0201] In some embodiments, the disease or condition is maltose sensitivity or lactose intolerance and the payload protein is maltase. In some embodiments, the disease or condition is maltose sensitivity and the payload protein is maltase. In some embodiments, the disease or condition is lactose intolerance and the payload protein is maltase.

[0202] In some embodiments, the disease or condition is pernicious anemia and the payload protein is intrinsic factor.

[0203] In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is lysozyme, nisin, a defensin, magainin, cateslytin, or any combination thereof. In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is lysozyme. In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is 54 IPTS / 200107384.1Attorney Matter No. TNZ-017WO nisin. In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is a defensing. In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is magainin. In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is cateslytin.

[0204] In some embodiments, the disease or condition is type 1 or type 2 diabetes mellitus and the payload protein is insulin, or an incretin. In some embodiments, the disease or condition is type 1 diabetes mellitus and the payload protein is insulin, or an incretin. In some embodiments, the disease or condition is type 1 diabetes mellitus and the payload protein is insulin. In some embodiments, the disease or condition is type 1 diabetes mellitus and the payload protein is an incretin. In some embodiments, the disease or condition is type 2 diabetes mellitus and the payload protein is insulin, or an incretin. In some embodiments, the disease or condition is type 2 diabetes mellitus and the payload protein is insulin. In some embodiments, the disease or condition is type 2 diabetes mellitus and the payload protein is an incretin.

[0205] In some embodiments, the disease or condition is a metabolic disorder and the payload protein is xylose-isomerase, fructose, or gluco-amylase. In some embodiments, the disease or condition is a metabolic disorder and the payload protein is xylose-isomerase. In some embodiments, the disease or condition is a metabolic disorder and the payload protein is fructose. In some embodiments, the disease or condition is a metabolic disorder and the payload protein is gluco-amylase.

[0206] In some embodiments, the disease or disorder is obesity and the payload protein is cholecystokinin or Lumenal cholecystokinin release factor (LCRF). In some embodiments, the disease or disorder is obesity and the payload protein is cholecystokinin. In some embodiments, the disease or disorder is obesity and the payload protein is LCRF.

[0207] In some embodiments, the disease or condition has an inflammatory component and the payload protein is IL-2, IL-10, IL-21, IL-22, TGFβ, or any combination thereof, or any mutants thereof.

[0208] Methods of Treating Invertase / Sucrase and / or Isomaltase Deficiency

[0209] An engineered yeast may be used, for example, to treat an enzyme deficiency such as a deficiency of invertase and / or isomaltase. Accordingly, in some embodiments a method of treating a sucrase / invertase and / or isomaltase deficiency in a subject in need thereof is provided, the method comprising orally administering to the subject one or both of 1) a therapeutically effective amount of an engineered yeast having a heterologous nucleic acid genomically integrated into one or more safe harbor sites that encodes a first recombinant polypeptide comprising invertase (or a pro-drug or active variant thereof) and a first synthetic signal peptide and 2) a therapeutically 55 IPTS / 200107384.1Attorney Matter No. TNZ-017WO effective amount of an engineered yeast having a heterologous nucleic acid genomically integrated into one or more safe harbor sites that encodes a recombinant polypeptide comprising isomaltase (or a pro-drug or active variant thereof) and a second synthetic signal peptide, thereby treating the invertase and / or isomaltase deficiency. In some embodiments, the engineered yeast is selected from the group comprising Saccharomyces cerevisiae and Saccharomyces boulardii) In some embodiments, the invertase and / or isomaltase deficiency may be secondary to a functional gut disorder, such as, but not limited to, irritable bowel syndrome, functional dyspepsia, functional vomiting, functional abdominal pain, functional constipation, and / or functional diarrhea.

[0210] In some embodiments, a method of treating a sucrase / invertase and / or isomaltase deficiency is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), having a heterologous nucleic acid genomically integrated into one or more safe harbor sites that encodes one or both of 1) a nucleic acid encoding a recombinant polypeptide comprising isomaltase and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide and 2) a nucleic acid encoding a recombinant polypeptide comprising invertase and one or both of a) a pre-protein signal peptide and b) a pro- protein signal peptide, thereby treating the deficiency.

[0211] In some embodiments, the sucrase / invertase and / or isomaltase deficiency may be, for example, congenital sucrase-isomaltase deficiency. In any embodiment where a subject has both a sucrase / invertase and isomaltase deficiency and it is desired to administer engineered yeast to express both enzymes, the same yeast strain may be used to express both enzymes or one yeast strain may be used to express invertase and another yeast strain may be used to express isomaltase. In some embodiments, administration of both enzymes is performed utilizing one yeast strain to express both enzymes.

[0212] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0213] Method of Treating Lactose Intolerance 56 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0214] In some embodiments, a method of treating a lactase deficiency or lactose-intolerance in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast having a heterologous nucleic acid genomically integrated into one or more safe harbor sites that encodes a recombinant polypeptide comprising lactase (or a pro-drug or active variant thereof) and a synthetic signal peptide, thereby treating lactase deficiency or lactose-intolerance. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0215] In some embodiments, a method of treating a lactase deficiency / lactose-intolerance is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii) having a heterologous nucleic acid genomically integrated into one or more safe harbor sites encoding a recombinant polypeptide comprising lactase and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby treating the deficiency.

[0216] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0217] Method of Treating Pancreatic Disorders

[0218] In some embodiments, a method of treating a pancreatic disorder, such as pancreatitis or exocrine pancreatic insufficiency, in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast having a heterologous nucleic acid genomically integrated into one or more safe harbor sites encoding a recombinant polypeptide comprising one or more of triacylglycerol lipase, colipase, alpha- amylase, trypsin, and chymotrypsin and a synthetic signal peptide, thereby treating the disorder. In some embodiments, the engineered yeast is genetically modified to express a recombinant polypeptide comprising triacylglycerol lipase and a synthetic signal peptide as provided for herein, and is effective for treating one or both of pancreatitis or exocrine pancreatic insufficiency. In 57 IPTS / 200107384.1Attorney Matter No. TNZ-017WO some embodiments, the engineered yeast is genetically modified to express a recombinant polypeptide comprising colipase and a synthetic signal peptide as provided for herein, and is effective for treating one or both of pancreatitis or exocrine pancreatic insufficiency. In some embodiments, the engineered yeast is genetically modified to express a recombinant polypeptide comprising alpha-amylase and a synthetic signal peptide as provided for herein, and is effective for treating one or both of pancreatitis or exocrine pancreatic insufficiency. In some embodiments, the engineered yeast is genetically modified to express a recombinant polypeptide comprising trypsin and a synthetic signal peptide as provided for herein, and is effective for treating one or both of pancreatitis or exocrine pancreatic insufficiency. In some embodiments, the engineered yeast is genetically modified to express a recombinant polypeptide comprising chymotrypsin and a synthetic signal peptide as provided for herein, and is effective for treating one or both of pancreatitis or exocrine pancreatic insufficiency.

[0219] In some embodiments, a method of treating pancreatitis or exocrine pancreatic insufficiency is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide, thereby treating the disorder.

[0220] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0221] Method of Treating Celiac Disease / Gluten Intolerance / Refractory Sprue

[0222] In some embodiments, a method of treating a deficiency of one or more of aspergillus niger prolyl endoprotease (An-PEP), Myoxococcus xanthus prolyl endopeptpidase (Mx-PEP), Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, 58 IPTS / 200107384.1Attorney Matter No. TNZ-017WO subtilisin, sedolisin, and larozotide (or a pro-drug or active variant thereof) and a synthetic signal peptide, thereby treating the deficiency. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii. In some embodiments, the recombinant polypeptide comprises An-PEP and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue. In some embodiments, the recombinant polypeptide comprises Mx-PEP and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue. In some embodiments, the recombinant polypeptide comprises Aspergillus tubigensis prolyl endopeptidase and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue. In some embodiments, the recombinant polypeptide comprises subtilisin and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue. In some embodiments, the recombinant polypeptide comprises sedolisin and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue. In some embodiments, the recombinant polypeptide comprises larozotide and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue.

[0223] In some embodiments, a method of treating one or more of Celiac Disease, gluten intolerance, and refractory sprue is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising 1) one or more of An- PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide and 2) one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby treating the disease or disorder.

[0224] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 59 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0225] Methods of Treating Enteropeptidase / Enterokinase Deficiency

[0226] Enterokinase or enteropeptidase deficiency is an autosomal recessive disorder characterized by severe protein malabsorption in early infancy and may be treated by an engineered yeast according to the present disclosure. Accordingly, in some embodiments, a method of treating enterokinase / enteropeptidase deficiency in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or both of enteropeptidase (enterokinase) and proenteropeptidase and a synthetic signal peptide, thereby treating the disorder. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0227] In some embodiments, a method of treating enterokinase or enteropeptidase deficiency is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising 1) one or both of enteropeptidase / enterokinase and proenteropeptidase and 2) one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby treating the disorder.

[0228] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0229] Methods of Treating Small Intestine Bacterial Overgrowth or a Bacterial Infection

[0230] In some embodiments, a method of treating bacterial infection or bacterial overgrowth in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a 60 IPTS / 200107384.1Attorney Matter No. TNZ-017WO recombinant polypeptide comprising 1) one or both of lysozyme and intestinal alkaline phosphatase and 2) a synthetic signal peptide, thereby treating the infection or overgrowth. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence; and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii. In some embodiments, the bacterial infection or overgrowth may include, but not be limited to, a small intestine bacterial overgrowth, which may be associated with diabetes, a C. difficile infection, and intestinal bacterial overgrowth associated with cystic fibrosis. In some embodiments, the bacterial infection may be caused by be any gram-positive or gram-negative bacteria, such as, but not limited to, an infection of Escherichia Coli (E. Coli), Clostridioides difficile, P. aeruginosa, Shigella, Salmonella, Vibrio cholera, or cryptosporidium.

[0231] In some embodiments, a method of treating a bacterial overgrowth or infection is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising 1) one or both of lysozyme and intestinal alkaline phosphatase and 2) and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby treating the infection or overgrowth.

[0232] In some embodiments, other antibacterial proteins that may be produced by an engineered yeast and therefore provide treatment for bacterial overgrowth or infection in a subject include human beta defensins, peptide antimicrobials of animal origin (e.g., magainin, dermaseptin, cateslytin), and peptide antimicrobials of microbe origin (e.g., nisin, sakacin). In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0233] In some embodiments, the method of treating a bacterial infection with an engineered yeast genetically modified to express lysozyme, as described herein, may further comprise administering an antibacterial agent in combination with the engineered yeast. For example, a bacterial infection 61 IPTS / 200107384.1Attorney Matter No. TNZ-017WO may be treated by administering a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising a synthetic signal peptide and lysozyme and a therapeutically effective amount of an antibacterial agent. In some embodiments, the antibacterial agent is selected from the group comprising quinupristin, piperacillin, penicillin, clarithromycin, nitrofurantoin, ciprofloxacin, telithromycin, metronidazole, levofloxacin, erythromycin, theophylline, gemifloxacin, tetracycline, azithromycin, delafloxacin, eravacycline, moxifloxacin, dalbavancin, amoxicillin, fidaxomicin, tigecycline, ceftriaxone, minocycline, rifapentine, clindamycin, ceftazidime, oritayancin, norfloxacin, doxycycline, cefuroxime, tobramycin, ceftibuten, gentamicin, cefotaxime, vancomycin, telavancin, daptomycin, cephalexin, fofomycin, tedizolid, aztreonam, nafcillin, phenytoin, ertapenem, cefazolin, isoniazid, doripenem, rifabutin, meropenem, linezolid, oflaxacin, cefoxitin, oxacillin, warfarin, neomycin, rifampin, cefepime, and digoxin. In some embodiments, the antibacterial agent can be administered by any route, such as oral, topical, intranasal, mucosal, otic, parenteral, or the like.

[0234] Methods of Treating Gastrointestinal Disorders

[0235] In some embodiments, a method of treating inflammatory gastrointestinal disorders in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising intestinal alkaline phosphatase and a synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence; and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii). In some embodiments, the inflammatory gastrointestinal disorder is selected from the group including, but not limited to, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and necrotizing enterocolitis.

[0236] In some embodiments, a method for treating an inflammatory gastrointestinal disorder is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising intestinal alkaline phosphatase and one or both of a) a pre- protein signal peptide and b) a pro-protein signal peptide, thereby treating the disorder. In some embodiments, the inflammatory gastrointestinal disorder is selected from the group comprising IBS, IBD, and necrotizing enterocolitis.

[0237] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount 62 IPTS / 200107384.1Attorney Matter No. TNZ-017WO of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0238] Methods of Treating Insulin Deficiency / Diabetes

[0239] An engineered yeast may be used to treat an insulin deficiency or disorder, such as type 1 and type 2 diabetes mellitus. Accordingly, in some embodiments, a method of treating type 1 or type 2 diabetes mellitus in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising insulin (or a peptide analog or pro-drug thereof) and a synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence as provided for herein and b) a pro-protein amino acid sequence as provided for herein. In some embodiments, the engineered yeast may be any strain as disclosed herein. . In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0240] In some embodiments, a method of treating an insulin deficiency / diabetes is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising insulin (or a peptide analog or pro-drug thereof) and one or both of a) a pre-protein signal peptide, and b) a pro-protein signal peptide, thereby treating the deficiency or disease.

[0241] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 63 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0242] In some embodiments, a method of treating type 1 or type 2 diabetes mellitus in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising an incretin and a synthetic signal peptide, thereby treating the type 1 or type 2 diabetes mellitus. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre- protein amino acid sequence; and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii. In some embodiments, the incretin is selected from the group including, but not limited to, GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin, diaglutide, exenatide, liraglutide, semaglutide, sitagliptin, saxagliptin, alogliptin, linagliptin, and GIP.

[0243] In some embodiments, a method of treating an insulin deficiency / diabetes is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising an incretin and one or both of a) a pre-protein signal peptide and b) a pro- protein signal peptide, thereby treating the deficiency or disease. In some embodiments, the incretin is selected from the group including, but not limited to, GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin, diaglutide, exenatide, liraglutide, semaglutide, sitagliptin, saxagliptin, alogliptin, linagliptin, and GIP).

[0244] Methods of Repairing GI Epithelium

[0245] An engineered yeast may be used to promote healing and repair of GI epithelium, for example, as caused by any disease or condition such as IBD or IBS, through the production of trefoil factors (e.g., TFF1 / 2 / 3) or IGF-1.

[0246] Accordingly, in some embodiments, a method of promoting growth and repair in GI endothelium in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of TFF1, TFF2, TFF3, or IGF-1 and synthetic signal peptide, thereby promoting growth and repair in GI endothelium. In some embodiments, the synthetic signal peptide comprises one or both of a) a pre-protein amino acid sequence; and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. . In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii. 64 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0247] In some embodiments, a method of promoting GI growth and repair is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising TFF1, TFF2, TFF3, or IGF-1 and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby promoting GI growth and repair.

[0248] In any embodiment, growth and / or repair of GI epithelium may be in the context of a condition or disease such as short bowel syndrome, IBS, IBD, or any other disease where the GI epithelium is damaged or dysfunctional. In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0249] Methods of Treating Short Bowel Syndrome

[0250] An engineered yeast may be used to treat short bowel syndrome. Accordingly, in some embodiments, a method of treating short bowel syndrome in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising IGF-1, GLP-2 or any synthetic analog or prodrug thereof and synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) a pre-protein amino acid sequence; and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0251] In some embodiments, a method of treating short bowel syndrome is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising IGF-1, GLP- 2 or any synthetic analog or prodrug thereof and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby treating short bowel syndrome. 65 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0252] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0253] Methods of Treating Trehalose Sensitivity

[0254] Trehalase deficiency is a metabolic condition where the body lacks the enzyme trehalase and is therefore unable to convert trehalose into glucose. Accordingly, in some embodiments, a method of treating a trehalase deficiency in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising trehalase (or a pro-drug or active variant thereof) and a synthetic signal peptide, thereby treating the deficiency. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. . In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0255] In some embodiments, a method of treating trehalose sensitivity is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising trehalase and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby treating the trehalose sensitivity.

[0256] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 66 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0257] Methods of Treating Pernicious Anemia

[0258] Pernicious anemia is a rare blood disorder characterized by the inability of the body to properly utilize vitamin B12, resulting from the lack of the gastric protein intrinsic factor, without which B12 cannot be absorbed. Accordingly, in some embodiments, a method of treating pernicious anemia in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising intrinsic factor (or a pro-drug or active variant thereof) and a synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein.. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0259] In some embodiments, a method of treating pernicious anemia is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising intrinsic factor and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide.

[0260] Method of Reducing Inflammation

[0261] An engineered yeast may be used to produce pro-repair cytokines such as IL-2, IL-10, IL- 21, IL-22, and / or TGFβ, and / or any combinations or mutants thereof, which may be suitable for treating a variety of diseases and conditions. Further, engineered yeast may be used to produce anti-TNFα antibodies or fragments of anti-TNFα antibodies. Oral administration of IL-2, IL-10, IL-21, IL-22, TGFβ and / or anti-TNFα antibodies or fragments thereof may be beneficial for treating and repairing damage caused by inflammatory GI conditions, such as IBS, IBD, and the like. In some embodiments, an engineered yeast genetically modified to express IL-10 may be orally administered to a subject to treat Crohn’s disease or inhibit tumor metastasis. Accordingly, in some embodiments, a method of treating an inflammatory condition in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of IL-2, IL-10, IL-21, IL-22, TGFβ, and anti-TNFα antibodies or fragments thereof, or an analog or prodrug thereof and synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence and b) a pro-protein amino 67 IPTS / 200107384.1Attorney Matter No. TNZ-017WO acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. . In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0262] In some embodiments, a method of treating inflammation is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising one or more of IL-2, IL-10, IL-21, IL-22, TGFβ, and anti-TNFα antibodies or fragments thereof, or an analog or prodrug thereof and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby treating the inflammation.

[0263] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0264] Method of Treating Cancer

[0265] An engineered yeast may be used for treating a variety of cancers, for example, but not limited to, cancers of the GI tract. Accordingly, in some embodiments, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of an anti-cancer therapeutic and synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre- protein amino acid sequence and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. . In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0266] In some embodiments, a method of treating cancer is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising one or more an anti- 68 IPTS / 200107384.1Attorney Matter No. TNZ-017WO cancer therapeutic and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby treating the cancer.

[0267] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0268] Immunotherapy for Colorectal Cancer

[0269] An engineered yeast may be used to produce cytokines such as IL-2 and IL-21, or any combinations or mutants thereof, which may be suitable for use as an immunotherapy for the treatment of colorectal cancer. Accordingly, in some embodiments, a method of treating colorectal cancer in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of IL-2 or IL-21, or an analog or prodrug thereof and synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein.. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0270] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0271] Method of Promoting Appetite Suppression 69 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0272] An engineered yeast may be used to induce the release of the peptide hormone cholecystokinin (CCK, also known as pancreozymin), which has important roles in digestion and satiety. Oral administration of luminal CCK-releasing factor (LCRF) may be beneficial for promoting appetite suppression, delaying of gastric emptying, and / or inducing pancreatic secretion. Other proteins that exhibit these same functions include casein and soy proteins. Thus, administration of LCRF, casein, and / or soy proteins may be useful in the treatment of several digestive disorders and obesity through i) the suppression of appetite and ii) the promotion of digestion. In some embodiments, an engineered yeast genetically modified to express LCRF, casein, and / or soy proteins may be orally administered to a subject to promote appetite suppression. Accordingly, in some embodiments, a method of promoting appetite suppression in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising LCRF and synthetic signal peptide. In some embodiments, the recombinant polypeptide comprises casein. In some embodiments, the recombinant polypeptide comprises soy proteins. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. . In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0273] In some embodiments, a method of promoting appetite suppression is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby promoting appetite suppression.

[0274] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges. 70 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0275] Method of Delaying of Gastric Emptying

[0276] An engineered yeast may be used to induce the release of the peptide hormone cholecystokinin (CCK, also known as pancreozymin), which has important roles in digestion and satiety. Oral administration of luminal CCK-releasing factor (LCRF) may be beneficial for promoting appetite suppression, delaying of gastric emptying, and / or inducing pancreatic secretion. Other proteins that exhibit these same functions include casein and soy proteins. Thus, administration of LCRF, casein, and / or soy proteins may be useful in the treatment of several digestive disorders and obesity through i) the suppression of appetite and ii) the promotion of digestion. In some embodiments, an engineered yeast genetically modified to express LCRF, casein, and / or soy proteins may be orally administered to a subject to promote appetite suppression. Accordingly, in some embodiments, a method of delaying of gastric emptying in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising LCRF and synthetic signal peptide. In some embodiments, the recombinant polypeptide comprises casein. In some embodiments, the recombinant polypeptide comprises soy proteins. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. . In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0277] In some embodiments, a method of delaying of gastric emptying is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby delaying gastric emptying.

[0278] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 71 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0279] Method of Inducing Pancreatic Secretion

[0280] An engineered yeast may be used to induce the release of the peptide hormone cholecystokinin (CCK, also known as pancreozymin), which has important roles in digestion and satiety. Oral administration of luminal CCK-releasing factor (LCRF) may be beneficial for promoting appetite suppression, delaying of gastric emptying, and / or inducing pancreatic secretion. Other proteins that exhibit these same functions include casein and soy proteins. Thus, administration of LCRF, casein, and / or soy proteins may be useful in the treatment of several digestive disorders and obesity through i) the suppression of appetite and ii) the promotion of digestion. In some embodiments, an engineered yeast genetically modified to express LCRF, casein, and / or soy proteins may be orally administered to a subject to promote appetite suppression. Accordingly, in some embodiments, a method of inducing pancreatic secretion in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising LCRF and synthetic signal peptide. In some embodiments, the recombinant polypeptide comprises casein. In some embodiments, the recombinant polypeptide comprises soy proteins. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. . In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0281] In some embodiments, a method of inducing pancreatic secretion is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising CCK or LCRF and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby inducing pancreatic secretion.

[0282] In some embodiments, a method of treating obesity is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising CCK or LCRF and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby treating the obesity. 72 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0283] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0284] Methods of Treating Metabolic Disorders

[0285] An engineered yeast may be used for treating a variety of metabolic disorders. Accordingly, in some embodiments, a method of treating a metabolic disorder in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of xylose-isomerase, fructose, gluco-amylase, and synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0286] In some embodiments, a method of treating a metabolic disorder is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising one or more of xylose-isomerase, fructose, or gluco-amylase and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby treating the metabolic disorder.

[0287] In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020CFUs, about 103to 1015CFUs, 104to 1010CFUs, or about 102to about 108CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103to about 1015CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103CFUs, or about 104CFUs to about 108CFUs, about 1010CFUs, about 73 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 1015CFUs, or about 1020CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.

[0288] Compositions of Engineered Yeast

[0289] In any method of administering the engineered yeast as a therapeutic, the engineered yeast may be incorporated into a composition suitable for oral administration to the subject. Accordingly, in some embodiments, a composition is provided, the composition comprising an engineered yeast as provided for herein. Advantageously, the engineered yeast, as disclosed herein, retain activity even after lyophilization and / or freeze-drying providing a particularly shelf-stable form for incorporating into pharmaceutical products, such as those for reconstitution prior to consumption. Accordingly, in some embodiments, the engineered yeast in the pharmaceutical composition can be provided in a lyophilized or freeze-dried form. An oral composition comprising an engineered yeast, as disclosed herein, may be in the form of a pill, tablet, capsule, microcapsule, powder, sachet, dragee, gel, liquid, suspension, solution, food product, cream or granule. In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutically acceptable excipient is selected from the group including, but not limited to, carriers, solvents, co-solvents, emulsifiers, lubricants, disintegrants, binders, fillers, glidants, rheology agents, solubilizers, antimicrobials, antioxidants, preservatives, colorants, flavor agents, emollients, pH modifiers, and the like.

[0290] In some embodiments, food products may include, but are not limited to, a dairy product, a yoghurt, an ice cream, a milk-based drink, a milk-based garnish, a pudding, a milkshake, an ice tea, a fruit juice, a diet drink, a soda, a sports drink, a powdered drink mixture for dietary supplementation, an infant and baby food, a calcium-supplemented orange juice, a sauce or a soup.

[0291] Agricultural Compositions and Methods of their Use

[0292] An engineered yeast may be used to produce agricultural payload proteins such as, but not limited to, decomposition enzymes (e.g., cellulose), soil and other agricultural enzymes (e.g., lipases, proteases, polymerases, amylases, peroxidases, catalases, beta glucosidase, FDA hydrolysis, amidase, urease, phosphatase, sulfatase) fungicides (e.g., chitinase, chitin-binding proteins, cyclophilin-like proteins, defensins, lipid transfer proteins, miraculin-like proteins, nucleases, thaumatin-like proteins, and the like), insecticides (e.g., Vip1, Vip2, Vip3, Cry proteins, and the like), plant activators (e.g., branched-β-glucans, chitin oligomers, pectolytic enzymes, elicitor activity independent from enzyme activity (e.g. endoxylanase, elicitins, PaNie), avr gene products (e.g., AVR4, AVR9), viral proteins (e.g., vial coat protein, Harpins), flagellin, protein or peptide toxin (e.g., victorin), glycoproteins, glycopeptide fragments of invertase, syringolids, Nod factors (lipochitoolingo-saccharides), FACs (fatty acid amino acid conjugates), ergosterol, bacterial toxins (e.g., coronatine), and sphinganine analogue mycotoxins (e.g., fumonisin B1), 74 IPTS / 200107384.1Attorney Matter No. TNZ-017WO which may be suitable for treating a variety of diseases and conditions. Application of one or more of the above described agricultural payload proteins to an agricultural environment, such as a crop, garden, or the like, may be beneficial for promoting soil and plant health. Accordingly, in some embodiments, a method of promoting soil and / or plant health is provided, the method comprising applying to the soil or plant an effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of an agricultural payload protein and synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence and b) a pro-protein amino acid sequence. In some embodiments, the engineered yeast may be any strain as disclosed herein.. In some embodiments, the engineered yeast is selected from the group comprising S. cerevisiae and S. boulardii.

[0293] In some embodiments, a method of promoting soil and / or plant health is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a heterologous nucleic acid genomically integrated into one or more safe harbor sites, the heterologous nucleic acid encoding a recombinant polypeptide comprising one or an agricultural payload protein as provided for herein and one or both of a) a pre-protein signal peptide and b) a pro-protein signal peptide, thereby promoting soil and / or plant health.

[0294] In some embodiments, administering may be performed via any route. In some embodiments, the composition is sprayed onto the soil and / or plants. The agriculturally effective amount of engineered yeast may be any amount necessary to result in the desired beneficial effect to soil and or plant health. ENUMERATED EMBODIMENTS:

[0295] In some embodiments, the following embodiments are provided: 1. An engineered yeast cell comprising a heterologous nucleic acid genomically integrated into one or more safe harbor sites. 2. The engineered yeast cell of embodiment 1, wherein the yeast cell is a yeast from the genus Saccharomyces. 3. The engineered yeast cell of embodiment 1 or 2, wherein the yeast cell is a Saccharomyces species selected from the group comprising Saccharomyces cerevisiae and Saccharomyces boulardii. 4. The engineered yeast cell of any one of embodiments 1 -3, wherein the yeast cell is the Saccharomyces species Saccharomyces boulardii. 5. The engineered yeast cell of embodiment 1, wherein the heterologous nucleic acid is integrated at about 3 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or 75 IPTS / 200107384.1Attorney Matter No. TNZ-017WO more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, about 100 or more, about 105 or more, about 110 or more, about 115 or more, about 120 or more, about 125 or more, about 130 or more, about 135 or more, about 140 or more, about 145 or more, or about 150 or more safe harbor sites. 6. The engineered yeast cell of embodiment 5 , wherein the heterologous nucleic acid is integrated at about 3 or more, about 5 or more, about 10 or more, about 15 or more, or about 20 or more safe harbor sites. 7. The engineered yeast cell of any one of embodiments 5-6, wherein the heterologous nucleic acid is integrated at about 10 or more or about 15 or more safe harbor sites. 8. The engineered yeast cell of any one of embodiments 5-7, wherein the heterologous nucleic acid is integrated at about 14 safe harbor sites. 9. The engineered yeast cell of any one of embodiments 1 -8, wherein the safe harbor sites include one or more safe harbor sites selected from the group comprising a long terminal repeat (LTR) of a transposable element (Ty element), a non-essential gene, a metabolic auxotrophic gene, and a transcriptionally inactive region in the genome. 10. The engineered yeast cell of embodiment 9, wherein the safe harbor sites include one or more safe harbor sites from a long terminal repeat (LTR) of a Ty element and a metabolic auxotrophic gene. 11. The engineered yeast cell of embodiment 9, wherein the one or more metabolic auxotrophic genes are selected from the group comprising LEU2 (YCL018W), HIS3 (YOR202W), and URA3 (YEL021W). 12. The engineered yeast cell of embodiment 11, wherein the metabolic auxotrophic gene comprises a 5’ insertion and a 3’ insertion point. 13. The engineered yeast cell of embodiment 12, wherein the 5’ insertion and a 3’ insertion point are selected from the group comprising the pairs 1-3: Pair Sequence 5’ of insertion point Sequence 3’ of insertion point AT A CT 6)76 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 14. The engineered yeast cell of embodiment 9, wherein each of the one or more LTRs of a Ty element are located in chromosome number 1, 2, 4, 5, 6, 7, 13, 14, 15, or 16 of Saccharomyces boulardii. 15. The engineered yeast cell of embodiment 14, wherein each of the one or more LTRs of a Ty element comprise a 5’ insertion and a 3’ insertion point. 16. The engineered yeast cell of embodiment 15, wherein the 5’ insertion and a 3’ insertion point are selected from the group comprising the pairs 4-15: Pair Sequence 5’ of insertion point Sequence 3’ of insertion point 4 CCGACTGCCATGCAATGTGCTTTTCTGGATC TCTTTTTCTTCTTTTCATTTTTAGAGGCGTT TCACTCATGATCATAATGG (SEQ ID NO: 7) TTTTCACTAATAGCTACAG (SEQ ID NO:19) A : A O: TC T AT : AG 5) T TC C O: T O: C :. e eng neere yeas ce o any one o em o men s - , w ere n e e ero ogous nucleic acid comprises a nucleic acid sequence that encodes a payload protein. 18. The engineered yeast cell of embodiment 17, wherein the payload protein is an enzyme, a growth factor, insulin, an incretin, a cytokine, an antibody or fragment thereof, an antimicrobial peptide, a mucosal protein, an agricultural product, a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, a nutritional protein, an antiviral, an enzyme inhibitor, or a hormone. 77 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 19. The engineered yeast cell of embodiment 18, wherein the enzyme is selected from the group comprising invertase, isomaltase, lactase, lysozyme, alkaline phosphatase, and An-PEP. 20. The engineered yeast cell of embodiment 19, wherein the enzyme is invertase. 21. The engineered yeast cell of embodiment 19, wherein the enzyme is alkaline phosphatase. 22. The engineered yeast cell of embodiment 21, wherein the alkaline phosphatase is human intestinal alkaline phosphatase and comprises a sequence of SEQ ID NO: 76. 23. The engineered yeast cell of embodiment 21, wherein the alkaline phosphatase is yeast alkaline phosphatase and comprises a sequence of SEQ ID NO: 75. 24. The engineered yeast cell of embodiment 18, wherein the incretin is selected from the group comprising GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, and nesfatin. 25. The engineered yeast cell of embodiment 18, wherein cytokine is Il-2, IL-10, IL-21, or IL-22. 26. The engineered yeast cell of embodiment 25, where the cytokine is IL-10 and comprises a sequence of any one of SEQ ID NO: 71 or 81-103. 27. The engineered yeast cell of embodiment 25, where the cytokine is IL-21 and comprises a sequence of SEQ ID NO: 72. 28. The engineered yeast cell of embodiment 25, where the cytokine is IL-22 and comprises a sequence of SEQ ID NO: 73, 74, or 104. 29. The engineered yeast cell of embodiment 25, where the cytokine is IL-2 and comprises a sequence of any one of SEQ ID NOs: 105-108. 30. The engineered yeast cell of embodiment 18, wherein the mucosal protein is selected from the group comprising trefoil factor, Reg3 protein, and superoxide dismutase. 31. The engineered yeast cell of embodiment 18, wherein the agricultural product is selected from the group comprising pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, and fertilizer. 32. The engineered yeast cell of any one of embodiments 1-31, wherein the heterologous nucleic acid comprises a signal peptide. 33. The engineered yeast cell of embodiment 31, wherein the signal peptide is as provided for in WO2022 / 192675. 34. The engineered yeast cell of embodiment 32, wherein the signal peptide comprises a pre- protein signal peptide, a pro-protein signal peptide, or a combination thereof. 35. The engineered yeast cell of embodiment 33, wherein the pre-protein signal peptide is selected from: MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33), 78 IPTS / 200107384.1Attorney Matter No. TNZ-017WO MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61), MKFKLTLLAAILAYANTVLVAS (SEQ ID NO:62), MKFKLTLLAALSALAALALAAP (SEQ ID NO:63), or MKFKLTIFAALLALAALALAAP (SEQ ID NO:64). 36. The engineered yeast cell of embodiment 33 or 34, wherein the pro-protein signal peptide is selected from: EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), or DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36). 37. The engineered yeast cell of any one of embodiments 33-35, wherein the signal peptide comprises: MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), 79 IPTS / 200107384.1Attorney Matter No. TNZ-017WO MKFKLTLLAAILAYANTVLVAS (SEQ ID NO: 62) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTLLAAILAYANTVLVAS (SEQ ID NO: 62) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKLTLLAAILAYANTVLVAS (SEQ ID NO: 62) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKFKLTLLAALSALAALALAAP (SEQ ID NO: 63) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTLLAALSALAALALAAP (SEQ ID NO: 63) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKLTLLAALSALAALALAAP (SEQ ID NO: 63) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKFKLTIFAALLALAALALAAP (SEQ ID NO: 64) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTIFAALLALAALALAAP (SEQ ID NO: 64) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKLTIFAALLALAALALAAP (SEQ ID NO: 64) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36). 38. The engineered yeast cell of embodiment 32 or 34, wherein the signal peptide comprises the sequence of MKFKLTLLAALLALAALVLAASEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASR HKRDV (SEQ ID NO: 37). 39. The engineered yeast cell of embodiment 32 or 34, wherein the signal peptide comprises the sequence of MKFKSALFAALLALAALVLAAPEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASR HKRDV (SEQ ID NO: 65). 40. The engineered yeast cell of embodiment 32 or 34, wherein the signal peptide comprises the sequence of MKFKLTLLAAILAYANTVLVASEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRH KRDV (SEQ ID NO: 66). 41. The engineered yeast cell of embodiment 32 or 34, wherein the signal peptide comprises the sequence of MKFKLTLLAALSALAALALAAPEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASR HKRDV (SEQ ID NO: 67). 80 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 42. The engineered yeast cell of embodiment 32 or 34, wherein the signal peptide comprises the sequence of MKFKLTIFAALLALAALALAAPEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRH KRDV (SEQ ID NO: 68). 43. The engineered yeast cell of any one of embodiments 1-38, wherein the heterologous nucleic acid comprises a homology arm at the 5’ end and a homology arm at the 3’ end of the heterologous nucleic acid. 44. The engineered yeast cell of embodiment 43, wherein the homology arm at the 5’ end and the homology arm at the 3’ end of the heterologous nucleic acid comprise a sequence complementary to the 5’ insertion site and 3’ insertion stie, respectively. 45. The engineered yeast cell of embodiment 43 or 44, wherein the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-3 and 7-18. 46. The engineered yeast cell of embodiment 43 or 44, wherein the homology arm at the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4-6 and 19-30. 47. The engineered yeast cell of any one of embodiments 1-46, wherein the heterologous nucleic acid comprises a nucleic acid sequence encoding an anchoring protein. 48. The engineered yeast cell of embodiment 47, wherein the anchoring protein is Sed1 or CWP2. 49. The engineered yeast cell of embodiment 47 or 48, wherein the anchoring protein is Sed1 and comprises a sequence of SEQ ID NO: 69. 50. The engineered yeast cell of embodiment 47 or 48, wherein the anchoring protein is CWP2 and comprises a sequence of SEQ ID NO: 70. 51. The engineered yeast cell of any one of embodiments 1-46, wherein the heterologous nucleic acid comprises a constitutive or inducible promoter. 52. The engineered yeast cell of embodiment 51, wherein the constitutive or inducible promoter is a TEF1 promoter or a TEF1 U3 promoter. 53. The engineered yeast cell of any one of embodiments 1-52, wherein the heterologous nucleic acid comprises one or two terminator sequences. 54. The engineered yeast cell of embodiment 53, wherein the one or two terminator sequences are a CYC1 terminator or a ADH1 terminator. 55. The engineered yeast cell of any one of embodiments 17-54, wherein the heterologous nucleic acid is integrated at 14 safe harbor sites and comprises a nucleic acid sequence that encodes invertase. 81 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 56. The engineered yeast cell of embodiment 55, wherein the heterologous nucleic acid comprises the following elements operably linked from 5’ to 3’: a CYC1 terminator sequence, a TEF1 promoter sequence, a Sb propeptide sequence, a sequence that encodes invertase, and a ADH1 terminator sequence. 57. The engineered yeast cell of any one of embodiments 1-56, further comprising a nuclease capable of generating a break in the 5’ integration site or 3’ integration site. 58. The engineered yeast cell of embodiment 57, wherein the nuclease is a nuclease selected from the group comprising TALEN, ZFN, a Cas protein, and HO endonuclease. 59. The engineered yeast cell of embodiment 58, wherein the nuclease is a Cas protein. 60. The engineered yeast cell of embodiment 58 or 59, wherein the nuclease is Cas9. 61. A method of making the engineered yeast cell of any one of embodiments 1-60 comprising targeted insertion of a heterologous nucleic acid into one or more safe harbor sites. 62. The method of embodiment 61, where the one or more safe harbor sites each comprise a 5’ integration site and a 3’ integration site. 63. The method of embodiment 61 or 62, comprising a nuclease capable of generating a break in the 5’ integration site or 3’ integration site. 64. The method of any one of embodiments 61-63, wherein the nuclease is a nuclease selected from the group comprising TALEN, ZFN, a Cas protein, and HO endonuclease. 65. The method of any one of embodiments 61-64, wherein the nuclease is a Cas protein. 66. The method of any one of embodiments 61-65, wherein the nuclease is Cas9. 67. A method for treating a disease or a condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the engineered yeast cell of any one of embodiments 1- 60. 68. The method of embodiment 67, wherein the disease or condition is selected from an infection, an autoimmune disease, primary (congenital) enzymatic deficiency, enzymatic deficiencies secondary to functional gut disorders, diabetes, obesity, a metabolic disorder, intestinal bacterial overgrowth, enteric infection, bacterial vaginosis, inflammatory bowel disease, irritable bowel syndrome, small bowel syndrome, Celiac disease, gluten intolerance, colitis, peptic ulcer, or another GI condition or disorder. 69. The method of embodiment 67 or 68, wherein the disease or condition is an enzyme deficiency and the payload protein is an enzyme. 70. The method of embodiment 67 or 68, wherein the disease or condition is congenital sucrase-isomaltase deficiency and the payload protein is one or both of invertase and isomaltase. 82 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 71. The method of embodiment 67 or 68, wherein the disease or condition is one or both of sucrose and isomaltase intolerance secondary to a functional gut disorder and the payload protein is one or both of invertase and isomaltase. 72. The method of embodiment 67 or 68, wherein the disease or condition is one or more of gluten intolerance, refractory sprue, or Celiac disease and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide. 73. The method of embodiment 67 or 68, wherein the disease or condition is pancreatitis or exocrine pancreatic insufficiency and the payload protein is selected from one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin. 74. The method of embodiment 67 or 68, wherein the disease or condition is enteropeptidase deficiency or enterokinase deficiency and the payload protein is one or all of enteropeptidase, proenteropeptidase, and enterokinase. 75. The method of embodiment 67 or 68, wherein the disease or condition is small intestinal bacterial overgrowth, inflammatory bowel disease, irritable bowel syndrome, C. difficile infection, cystic fibrosis, necrotizing enterocolitis, and diabetes, and the payload protein is intestinal alkaline phosphatase. 76. The method of embodiment 67 or 68, wherein the disease or condition is caused by, or present with, endotoxemia, and the payload protein is human alkaline phosphatase, yeast alkaline phosphatase, bovine alkaline phosphatase, Cobetia amphilecti alkaline phosphatase, Paernibacillus lentus alkaline phosphatase, human lactoferrin (LF-22), human cathelicidin (LL- 37), human β-defensin-3 (HβD-3), human α-defensin 5 (HD5), human acyloxyacyl hydrolase (AOAH), Limulus polyphemus anti-lipopolysaccharide factor (ALF-L), tachypleus plasma lectin 2 (TPL2), human bactericidal permeability-increasing protein (BPI), and Crassostrea gigas bactericidal permeability-increasing protein (CgBPI). 77. The method of embodiment 76, wherein the disease or condition is aging, anxiety, autism, Alzheimer’s disease, Amyotrophic lateral sclerosis, atherosclerosis, chronic fatigue syndrome, chronic heart disease, cirrhosis, depression, diabetes, exercise-induced enteropathy, HIV enteropathy, metabolic endotoxemia, NSAID enteropathy, nonalcoholic fatty liver disease, inflammatory bowel disease, irritable bowel syndrome, obesity, Parkinson’s disease, sepsis, small intestinal bacterial overgrowth, rheumatoid arthritis, or thrombosis. 78. The method of embodiment 67 or 68, wherein the disease or condition is short bowel syndrome and the payload protein is IGF-1, GLP-2, or a synthetic derivative of GLP-2. 79. The method of embodiment 67 or 68, wherein the disease or condition is lactose sensitivity or lactose intolerance and the payload protein is lactase. 83 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 80. The method of embodiment 67 or 68, wherein the disease or condition is trehalose sensitivity or lactose intolerance and the payload protein is trehalase. 81. The method of embodiment 67 or 68, wherein the disease or condition is maltose sensitivity or lactose intolerance and the payload protein is maltase. 82. The method of embodiment 67 or 68, wherein the disease or condition is pernicious anemia and the payload protein is intrinsic factor. 83. The method of embodiment 67 or 68, wherein the disease or condition is bacterial overgrowth and the payload protein is lysozyme, nisin, a defensin, magainin, cateslytin, or any combination thereof. 84. The method of embodiment 67 or 68, wherein the condition is a bacterial infection caused by one or more of E. coli, C. difficile, vibrio cholera, Shigella, Salmonella, Cryptosporidium, or any combination thereof. 85. The method of embodiment 67 or 68, wherein the condition is a viral infection. 86. The method of embodiment 67 or 68, wherein the disease or condition is type 1 or type 2 diabetes mellitus and the payload protein is insulin, or an incretin. 87. The method of embodiment 67 or 68, wherein the administering is oral or topical. 88. The method of embodiment 67 or 68, wherein the disease or condition has an inflammatory component and the payload protein is IL-2, IL-10, IL-22, TGFβ, an anti-TNFα antibody or fragment thereof, or any combination thereof. 89. The method of embodiment 67 or 68, wherein the disease or condition has an inflammatory component and the payload protein is IL-10. 90. The method of embodiment 67 or 68, wherein the disease or condition has an inflammatory component and the payload protein is IL-21. 91. The method of embodiment 67 or 68, wherein the disease or condition has an inflammatory component and the payload protein is IL-22. 92. The method of embodiment 67 or 68, wherein the disease or condition has an inflammatory component and the payload protein is IL-2. EXAMPLES

[0296] Example 1: Engineered yeast comprising a heterologous nucleic acid

[0297] Yeast cell strains were engineered to comprise a heterologous nucleic sequence encoding invertase integrated at more than one safe harbor site and shown to secrete a higher amount of invertase compared to a yeast strain with invertase on a plasmid that is not genomically integrated.

[0298] S. boulardii cells were engineered for stable and reliable expression of invertase by integrating copies of a heterologous nucleic sequence encoding invertase, an exemplary construct 84 IPTS / 200107384.1Attorney Matter No. TNZ-017WO of which is shown in Figure 1 into the S. boulardii genome. Multiple loci in the S boulardii genome were used as targets for genomic integration of the invertase expression construct and were targeted using a CRISPR-Cas9 mediated approach. The target loci along with the genomic coordinates of insertions are listed in Table 1. Figure 2 summarizes at which integration sites the invertase construct was integrated in the different engineered strains.

[0299] The target loci in Table 1 were chosen after surveying the genome for sites that were amenable for genetic manipulation, afforded advantages such as biocontainment, genetic stability of integrated alleles, high expression of integrated alleles, and would have low interference due to chromatin related positional effects and epigenetic effects.

[0300] Some of the selected target loci included the genes LEU2 (YCL018W), HIS3 (YOR202W), and URA3 (YEL021W) that code for enzymes required for the biosynthesis of leucine, histidine, and uracil respectively. The disruption of one or more of these genes results in one or more metabolic auxotrophies in the engineered strain and limits the growth of the strain to environments that have an exogenous supply of leucine, histidine, and / or uracil. This prevents the engineered strain from surviving for long periods in environments where the one or more metabolites are not available, potentially rendering it bio-contained.

[0301] Long terminal repeat (LTR) of the Ty elements in the yeast genome were additional selected target sites. LTR integration sites were chosen by manually screening the Saccharomyces genome database (www.yeastgenome.org) for LTR sites using the following criteria for selection: • Distance from centromeres and telomeres, for example, at least 20,000 base pairs away from a telomere and / or centromere. As these regions are generally silenced, and exhibit lowered transcriptional activity, they were avoided as target integration sites. • No neighboring essential genes to avoid any unwanted fitness defects in the strain. • The DNA sequence of a chosen LTR sequence was then used to search for similar sequences in the S. boulardii genome. LTR sequences can have high homology. To target unique sequences, the S. boulardii Ty LTR sequence was used to probe its suitability for site specific targeting using CRISPR while avoiding off target integrations. The gRNAs that were designed to target a selected site were required to be at least 3 nucleotide different from the next closest sequence.

[0302] An effort was also made to chose selected integration sites from different chromosomes to avoid potential stability effects due to recombination.

[0303] Each integration site was tested for integration with an initial CRISPR based single-site targeting experiment, and then the engineered yeast was used for further integrations at 85 IPTS / 200107384.1Attorney Matter No. TNZ-017WO additional sites with CRISPR based single-site targeting or multi-site targeting approaches as needed. Exemplary gRNA sequences that were used to integrate the heterologous nucleic acid at the indicated genes in the engineered yeast strains in Figure 2A are summarized in Table 5A below: Table 5A. gRNA sequences for targeted integration at the indicated genes Gene at integration site -S. Chr r vi i RNA2 (if li bl ) (5’t 3’ A AGene at gRNA1 (5’to 3’ sequence) gRNA2 (5’to 3’ sequence) inte ration site G

[0304] This approach of integrating at multiple LTR sites has not been performed in the probiotic strain S. boulardii. Previous approaches published in S. cerevisiae (Sakai et al.1990, Shi et al.2016, Huang et al.2020) rely on the similarity in sequence between LTRs as a strategy to randomly incorporate multiple copies of a DNA sequence in the yeast genome. Random insertion requires tedious methods to identify insertion sites and the copy number of insertions. 86 IPTS / 200107384.1Attorney Matter No. TNZ-017WO Further, compared to our approach, random insertion strategies generally have lower copy numbers as well as low expression due to the lack of site optimization. Thus, the approach of site selection optimizes for maximal expression of a payload with careful probiotic strain engineering strategies.

[0305] The engineered S. boulardii strains with integrations at a variety of genomic sites were assayed for production of the invertase enzyme. The amount of invertase present in engineered S. boulardii cells was quantified and compared with invertase activity of a wild-type S. boulardii strain. Results, depicted in Figure 3, show that an engineered S. boulardii strain shows increase in invertase activity with increasing number of stably integrated invertase constructs. An S. boulardii strain with 28 genomic copies of the expression cassette resulted in a 1,400% increase in invertase expression compared to a wild-type S. boulardii strain and a 180% increase in invertase expression compared to a S. boulardii strain with an extra-chromosomal plasmid that encoded invertase (Figure 4).

[0306] Further, results depicted in Figure 4 show that a S. boulardii strain with stably integrated invertase construct exhibited a 1,400% more secretion of invertase compared to a wild type S. boulardii strain and 300% more secretion of invertase compared to a strain carrying the invertase construct on a plasmid. Thus, these targeted genomic manipulations allowed for generation of stable S. boulardii strains that secrete higher amounts of invertase than a strain carrying the invertase expression system on a non-integrated plasmid. Further, another advantage of a genomically integrated sequence expressing invertase compared to a plasmid is that plasmid copy numbers can change with each cell division, which can lead to wide variation in expression of a payload protein, such as invertase, and hence could lead to unreliable expression of a payload protein. Example 2: Use of engineered yeast to generate yeast comprising additional payload proteins of interest.

[0307] The engineered yeast generated in Example 1 are used as a template organism to quickly produce yeast with other target payload proteins genomically integrated in the same safe harbor sites. Using gRNA specific to invertase (FIGs.2B and 2C), the invertase is removed from the genomic integration site and additional payload proteins, such as those provided for herein, are inserted. Integration of the alternate payload proteins is confirmed via standard methods. Example 3: Engineered yeast comprising a heterologous nucleic acid

[0308] Yeast cell strains are engineered to comprise a heterologous nucleic sequence encoding a payload protein, as disclosed herein, integrated at more than one safe harbor site and shown to 87 IPTS / 200107384.1Attorney Matter No. TNZ-017WO secret a higher amount of the payload protein, compared to a yeast with the payload protein on a plasmid that is not genomically integrated.

[0309] S. boulardii cells are engineered for stable and reliable expression of a payload protein by integrating copies of a heterologous nucleic sequence encoding the payload protein into the S. boulardii genome. Multiple loci in the S boulardii genome are used as targets for genomic integration of the payload protein expression construct and are targeted using a genomic editing system, such as ZFN, TALENS, or a CRISPR-Cas9 mediated approach.

[0310] The target loci are chosen after surveying the genome for sites that are amenable for genetic manipulation, afford advantages such as biocontainment, genetic stability of integrated alleles, high expression of integrated alleles, and have low interference due to chromatin related positional effects and epigenetic effects.

[0311] Some of the selected target loci may include the genes LEU2 (YCL018W), HIS3 (YOR202W), and URA3 (YEL021W) that code for enzymes required for the biosynthesis of leucine, histidine, and uracil respectively. The disruption of one or more of these genes can result in one or more metabolic auxotrophies in the engineered strain and limit the growth of the strain to environments that have an exogenous supply of leucine, histidine, and / or uracil. This would prevent the engineered strain from surviving for long periods in environments where the one or more metabolites are not available, potentially rendering it bio-contained.

[0312] Long terminal repeat (LTR) of the Ty elements in the yeast genome are also selected as target sites. LTR integration sites can be manually screened in the Saccharomyces genome database (www.yeastgenome.org) for LTR sites using the selection criteria discussed in Example 1.

[0313] Integration sites can be chosen from different chromosomes to avoid potential stability effects due to recombination.

[0314] Each integration site is tested for integration and then used for further integrations using a genomic editing system with subsequent single-site targeting as needed or multi-site targeting approaches.

[0315] The engineered S. boulardii strains with integrations at a variety of genomic sites are assayed for production of the payload protein. The amount of the payload protein present in engineered S. boulardii cells can be quantified with methods and assays known to the skilled person in the art, such as, for example, protein purification, activity assays depending on the identity of the payload protein, and / or immunoblot assays (e.g., Western Blot, ELISA, FACS). Results will show that engineered S. boulardii strains show increase in the amount and / or activity of the payload protein with increasing number of stably integrated constructs. 88 IPTS / 200107384.1Attorney Matter No. TNZ-017WO

[0316] Further, a S. boulardii strain with stably integrated payload protein constructs will exhibit more secretion of the payload protein as compared to a wild type S. boulardii strain or as compared to a strain carrying the payload protein construct on a plasmid. Thus, these targeted genetic manipulations allow for generation of stable S. boulardii strains that secrete higher amounts of the payload protein compared to a strain carrying the payload protein expression system on a plasmid. Further, another advantage of a genomically integrated sequence expressing the payload protein compared to a plasmid encoding the payload protein is that plasmid copy numbers may change with each cell division, which leads to wide variation in expression of a payload protein and can lead to unreliable expression of a payload protein. Example 4: Engineered yeast comprising a heterologous nucleic acid

[0317] Yeast cell strains are engineered to comprise a heterologous nucleic sequence encoding a payload protein integrated at more than one safe harbor site and secrete a higher amount of the payload protein compared to a yeast strain with the payload protein on a plasmid that is not genomically integrated.

[0318] S. boulardii cells are engineered for stable and reliable expression of a payload protein, e.g., a cytokine, such as IL-10, IL-21, or IL-22, or human intestinal alkaline phosphatase (hIAP) or yeast alkaline phosphatase (yAP), by integrating copies of a heterologous nucleic sequence into the S. boulardii genome. Exemplary constructs encoding IL-10, IL-21, or IL-22 are shown in Figures 5-8. Exemplary constructs encoding human IL2 and mutants thereof are shown in Figure 9. Exemplary constructs encoding mutant IL10, IL22, or combinations thereof are shown in FIG.10. Exemplary constructs encoding human intestinal alkaline phosphatase (hIAP) or yeast alkaline phosphatase (yAP) are shown in Figures 11(A, B, and C)-12(A, B, and C). In the exemplary constructs, “Sb v1” comprises or consists of SEQ ID NO: 31, “Sb v2” comprises or consists of SEQ ID NO: 61, “Sb v3” comprises or consists of SEQ ID NO: 62; “Sb v4” comprises or consists of SEQ ID NO: 63, “Sb v5” comprises or consists of SEQ ID NO: 64; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34.

[0319] The exemplary heterologous nucleic sequences shown in Figure 5-12 also comprise a promoter. The promoter is a TEF1 promoter or TEF1 U3 promoter. Exemplary sequences of these promoters are shown in Table 6. Table 6. SEQ ID NO Nucleic acid Sequence Description erIPTS / 200107384.1Attorney Matter No. TNZ-017WO ccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccactt caaaacacccaagcacagcatactaaatttcccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttgga 78 aaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaat TEF1 U3 romoterhe payload protein (e.g., cytokine IL-10, IL-21, IL-22, hIAP, or yAP) expression construct and are targeted using a CRISPR-Cas9 mediated approach. The target loci along with the genomic coordinates of insertions are listed in Table 1.

[0321] The target loci in Table 1 were chosen after surveying the genome for sites that were amenable for genetic manipulation, afforded advantages such as biocontainment, genetic stability of integrated alleles, high expression of integrated alleles, and would have low interference due to chromatin related positional effects and epigenetic effects.

[0322] Selected target loci can include the genes LEU2 (YCL018W), HIS3 (YOR202W), and URA3 (YEL021W) that code for enzymes required for the biosynthesis of leucine, histidine, and uracil respectively. The disruption of one or more of these genes results in one or more metabolic auxotrophies in the engineered strain and limits the growth of the strain to environments that have an exogenous supply of leucine, histidine, and / or uracil. This prevents the engineered strain from surviving for long periods in environments where the one or more metabolites are not available, potentially rendering it bio-contained.

[0323] Long terminal repeat (LTR) of the Ty elements in the yeast genome are additional selected target sites. LTR integration sites can be chosen by manually screening the Saccharomyces genome database (www.yeastgenome.org) for LTR sites using the selection criteria discussed in Example 1.

[0324] Selected integration sites from different chromosomes can be chosen to avoid potential stability effects due to recombination.

[0325] Each integration site is tested for integration with an initial CRISPR based single-site targeting experiment, and then the engineered yeast was used for further integrations at additional sites with CRISPR based single-site targeting or multi-site targeting approaches as needed. Exemplary gRNA sequences that are used to integrate a heterologous nucleic acid at the indicated genes in the engineered yeast strains are summarized in Table 5A and Table 5B.

[0326] The engineered S. boulardii strains with integrations at a variety of genomic sites are assayed for production of the payload protein (e.g., a cytokine or other payload protein described herein). The amount of payload protein (e.g., a cytokine) present in engineered S. boulardii cells is quantified and compared with amount of payload protein (e.g., a cytokine or other payload protein described herein) of a wild-type S. boulardii strain. Results show that an engineered S. 90 IPTS / 200107384.1Attorney Matter No. TNZ-017WO boulardii strain secretes a greater amount or higher percent of the payload protein (e.g., a cytokine or other payload protein described herein) with increasing number of stably integrated payload protein constructs. An S. boulardii strain with more genomic copies of the expression cassette results in a greater increase in payload protein (e.g., a cytokine or other payload protein described herein) expression as compared to a S. boulardii strain with an extra-chromosomal plasmid that encoded invertase. Example 5: Engineered yeast comprising a heterologous nucleic acid

[0327] Yeast cell strains are engineered to comprise a heterologous nucleic sequence encoding a payload protein and an anchoring protein integrated at more than one safe harbor site and secrete a higher amount of the payload protein that is anchored to the yeast cell surface compared to a yeast strain with the payload protein on a plasmid that is not genomically integrated.

[0328] S. boulardii cells are engineered for stable and reliable expression of a payload protein (e.g., an alkaline phosphatase, human intestinal alkaline phosphatase (hIAP), yeast alkaline phosphatase (yAP), or other payload protein described herein) with an anchoring protein, by integrating copies of a heterologous nucleic sequence encoding a payload protein, e.g., hIAP or yAP. Exemplary constructs encoding a payload protein, such as hIAP or yAP, are shown in Figures 11-12. In the exemplary constructs, “Sb v1” comprises or consists of SEQ ID NO: 31, “Sb v2” comprises or consists of SEQ ID NO: 61, “Sb v3” comprises or consists of SEQ ID NO: 62; “Sb v4” comprises or consists of SEQ ID NO: 63, “Sb v5” comprises or consists of SEQ ID NO: 64; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34. The role of different signal peptides is tested: constructs are made that include different pre- and / or pro-protein sequences.

[0329] The performance of each variant construct to secrete a payload, e.g., an alkaline phosphatase or other payload protein described herein, is assessed by performing an activity assay, e.g., a phosphatase assay, on culture supernatants. Briefly, S. boulardii strains carrying plasmids with variant synthetic signal sequences are grown for 24 hours in standard growth conditions. The cell culture supernatant is incubated with the substrate pNPP (p-Nitrophenyl phosphate) in buffer (250 mM Tris pH 8.8, 10 mM MgCl2, 1mM ZnCl2). A standard curve is setup using a purified payload protein, e.g., purified alkaline phosphatase enzyme, purchased commercially and used to estimate the activity of the payload protein, e.g., alkaline phosphatase or other payload protein described herein, from supernatants or cells. The change in color due to release of phosphate is measured kinetically at 405 nm and then is expressed as normalized yields by dividing units of phosphatase (µmol / min) by the number of CFUs, which is estimated based upon the corresponding culture’s optical density at 600 nm. The engineered S. boulardii strains can express and anchor the payload protein, e.g., hIAP, yAP, or other payload protein described herein, with an anchoring protein to the yeast cell surface. The engineered S. boulardii 91 IPTS / 200107384.1Attorney Matter No. TNZ-017WO strains expressing constructs with a combination of a pre- and -pro protein signal sequence have an increased level of the payload protein, e.g., hAIP or yAP or other payload protein described herein, that is anchored to the yeast cell surface as compared to strains expressing a construct that does not comprise a pre- and / or pro-protein signal sequence. Example 6: Engineered yeast comprising a yAP nucleic acid

[0330] S. boulardii cells were engineered to have two copies of yeast alkaline phosphatase (yAP) genomically integrated for stable and reliable expression. In a first assay, a cassette designed for expression of anchored yAP (construct yAP#16 as shown in FIG.12C and FIG. 13A comprising an anchoring protein Sed1) was inserted at the YEL021W location via CRISPR- Cas9. In a second assay, a cassette designed for expression of secreted yAP (construct yAP#6 as shown in FIG.12A) was inserted at the YEL021W location via CRISPR-Cas9. In both cassettes the “Sb v5” comprises or consists of SEQ ID NO: 64; “Sb propeptide 1” comprises or consists of SEQ ID NO: 34.

[0331] Briefly, the S. boulardii strain with the invertase allele as shown in FIG.2A integrated at several genomic locations was used to generate strains with two copies of the alkaline phosphatase gene integrated into the genome. Guide RNAs (see, e.g., FIG.2B) were designed to target the invertase gene to generate cut sites for insertion of the yAP gene alone for the secreted yAP construct or the yAP gene and the Sed1 gene for the anchored yAP construct. S. boulardii strains containing non-genomically integrated plasmids with the yAP#16 and yAP#6 constructs were used as controls.

[0332] The performance of each engineered S. boulardii strain to express yeast alkaline phosphatase was assessed by performing a phosphatase assay on cells or culture supernatants. Briefly, the engineered S. boulardii strains and the control plasmid bearing strains were grown for 24 hours in standard growth conditions. For the secreted yAP construct, the cell culture supernatant was incubated with the substrate pNPP (p-Nitrophenyl phosphate) in buffer (250 mM Tris pH 8.8, 10 mM MgCl2, 1mM ZnCl2). For the anchored yAP construct, the cells were lysed with lysis buffer, the cell lysate was centrifuged, and the resulting supernatant was incubated with the substrate pNPP in buffer. A standard curve was setup using purified alkaline phosphatase enzyme. The AP activity was assessed as described above in Example 4.

[0333] As shown in FIG.13B, the engineered S. boulardii strain with the genomically integrated anchored yAP construct (2x-yAP#16) showed about two-fold more yAP expression as compared to the S. boulardii with the plasmid-bearing strain (yAP#16). Thus, integration of anchored yAP resulted in increased expression compared to its expression from an episomal vector. In addition, the engineered S. boulardii strain with the genomically integrated secreted yAP (2x-yAP#6) 92 IPTS / 200107384.1Attorney Matter No. TNZ-017WO showed comparable expression of yAP in the supernatant as compared to the plasmid-bearing strain (yAP#6) (FIG.13C).

[0334] Furthermore, the S. boulardii with the genomically integrated gene does not require the use of antibiotics for growth and maintenance of the plasmid to express the payload protein. This strategy thus provides a simple method to generate a panel of S. boulardii strains for any protein or nucleic acid payload, whereby each strain has a different copy number of the expression cassette. This allows for fine-tuning the expression and / or secretion of a protein or nucleic acid payload to a requisite therapeutic level. Example 7: Method of treating diseases or conditions with engineered yeast comprising a heterologous nucleic acid

[0335] A subject in need thereof is administered an effective amount of an engineered yeast cell comprising a heterologous nucleic acid genomically integrated into one or more safe harbor sites as disclosed herein. Any of the heterologous nucleic acids discloses herein can be integrated into the engineered yeast cell. The subject can be suffering from an infection, an autoimmune disease, primary (congenital) enzymatic deficiency, enzymatic deficiencies secondary to functional gut disorders, diabetes, obesity, a metabolic disorder, intestinal bacterial overgrowth, enteric infection, bacterial vaginosis, inflammatory bowel disease, irritable bowel syndrome, small bowel syndrome, Celiac disease, gluten intolerance, colitis, peptic ulcer, or another GI condition or disorder. Alternatively, the subject can be suffering from a disease or condition caused by, or present with, endotoxemia, such as, but not limited to, aging, anxiety, autism, Alzheimer’s disease, Amyotrophic lateral sclerosis, atherosclerosis, chronic fatigue syndrome, chronic heart disease, cirrhosis, depression, diabetes, exercise-induced enteropathy, HIV enteropathy, metabolic endotoxemia, NSAID enteropathy, nonalcoholic fatty liver disease, inflammatory bowel disease, irritable bowel syndrome, obesity, Parkinson’s disease, sepsis, small intestinal bacterial overgrowth, rheumatoid arthritis, or thrombosis. An improvement in the disease or condition is observed.

[0336] It should be recognized that illustrated embodiments are only examples of the disclosed product and methods and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims. 93 IPTS / 200107384.1

Claims

Attorney Matter No. TNZ-017WO CLAIMS WHAT IS CLAIMED IS:

1. An engineered yeast cell comprising a heterologous nucleic acid genomically integrated into one or more safe harbor sites.

2. The engineered yeast cell of claim 1, wherein the yeast cell is a yeast from the genus Saccharomyces.

3. The engineered yeast cell of claim 1 or 2, wherein the yeast cell is a Saccharomyces species selected from the group comprising Saccharomyces cerevisiae and Saccharomyces boulardii.

4. The engineered yeast cell of any one of claims 1 -3, wherein the yeast cell is the Saccharomyces species Saccharomyces boulardii.

5. The engineered yeast cell of claim 1, wherein the heterologous nucleic acid is integrated at about 3 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, about 100 or more, about 105 or more, about 110 or more, about 115 or more, about 120 or more, about 125 or more, about 130 or more, about 135 or more, about 140 or more, about 145 or more, or about 150 or more safe harbor sites.

6. The engineered yeast cell of claim 5 , wherein the heterologous nucleic acid is integrated at about 3 or more, about 5 or more, about 10 or more, about 15 or more, or about 20 or more safe harbor sites.

7. The engineered yeast cell of any one of claims 5-6, wherein the heterologous nucleic acid is integrated at about 10 or more or about 15 or more safe harbor sites.

8. The engineered yeast cell of any one of claims 5-7, wherein the heterologous nucleic acid is integrated at about 14 safe harbor sites.

9. The engineered yeast cell of any one of claims 1 -8, wherein the safe harbor sites include one or more safe harbor sites selected from the group comprising a long terminal repeat (LTR) of a transposable element (Ty element), a non-essential gene, a metabolic auxotrophic gene, and a transcriptionally inactive region in the genome.

10. The engineered yeast cell of claim 9, wherein the safe harbor sites include one or more safe harbor sites from a long terminal repeat (LTR) of a Ty element and a metabolic auxotrophic gene. 94 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 11. The engineered yeast cell of claim 9, wherein the one or more metabolic auxotrophic genes are selected from the group comprising LEU2 (YCL018W), HIS3 (YOR202W), and URA3 (YEL021W).

12. The engineered yeast cell of claim 11, wherein the metabolic auxotrophic gene comprises a 5’ insertion and a 3’ insertion point.

13. The engineered yeast cell of claim 12, wherein the 5’ insertion and a 3’ insertion point are selected from the group comprising the pairs 1-3: Pair Sequence 5’ of insertion point Sequence 3’ of insertion point AT A CT 6) 1element are located in chromosome number 1, 2, 4, 5, 6, 7, 13, 14, 15, or 16 of Saccharomyces boulardii.

15. The engineered yeast cell of claim 14, wherein each of the one or more LTRs of a Ty element comprise a 5’ insertion and a 3’ insertion point.

16. The engineered yeast cell of claim 15, wherein the 5’ insertion and a 3’ insertion point are selected from the group comprising the pairs 4-15: Pair Sequence 5’ of insertion point Sequence 3’ of insertion point 4 CCGACTGCCATGCAATGTGCTTTTCTGGATC TCTTTTTCTTCTTTTCATTTTTAGAGGCGTT 9) A : A O: TC T AT : AG 5)IPTS / 200107384.1Attorney Matter No. TNZ-017WO Pair Sequence 5’ of insertion point Sequence 3’ of insertion point 11 TTGTTTATAAAAGCTATGAACTTAGGTCTAC ATTAATCATCTATTGGTGTGTACTCATACT AGAATGTACCAAAAGTTAT (SEQ ID NO 14) ACTTGTATCATATACAGTGT (SEQ ID NO: TC C O: T O: C :leic acid comprises a nucleic acid sequence that encodes a payload protein.

18. The engineered yeast cell of claim 17, wherein the payload protein is an enzyme, a growth factor, insulin, an incretin, a cytokine, an antibody or fragment thereof, an antimicrobial peptide, a mucosal protein, an agricultural product, a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, a nutritional protein, an antiviral, an enzyme inhibitor, or a hormone.

19. The engineered yeast cell of claim 18, wherein the enzyme is selected from the group comprising invertase, isomaltase, lactase, lysozyme, alkaline phosphatase, and An-PEP.

20. The engineered yeast cell of claim 19, wherein the enzyme is invertase.

21. The engineered yeast cell of claim 19, wherein the enzyme is alkaline phosphatase.

22. The engineered yeast cell of claim 21, wherein the alkaline phosphatase is human intestinal alkaline phosphatase and comprises a sequence of SEQ ID NO:

76.

23. The engineered yeast cell of claim 21, wherein the alkaline phosphatase is yeast alkaline phosphatase and comprises a sequence of SEQ ID NO:

75.

24. The engineered yeast cell of claim 18, wherein the incretin is selected from the group comprising GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, and nesfatin.

25. The engineered yeast cell of claim 18, wherein cytokine is IL-2, IL-10, IL-21, or IL-22.

26. The engineered yeast cell of claim 25, where the cytokine is IL-10 and comprises a sequence of any one of SEQ ID NO: 71 or 81-103.

27. The engineered yeast cell of claim 25, where the cytokine is IL-21 and comprises a sequence of SEQ ID NO:

72.

28. The engineered yeast cell of claim 25, where the cytokine is IL-22 and comprises a sequence of SEQ ID NO: 73, 74, or 104. 96 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 29. The engineered yeast cell of claim 25, where the cytokine is IL-2 and comprises a sequence of any one of SEQ ID NOs: 105-108 30. The engineered yeast cell of claim 18, wherein the mucosal protein is selected from the group comprising trefoil factor, Reg3 protein, and superoxide dismutase.

31. The engineered yeast cell of claim 18, wherein the agricultural product is selected from the group comprising pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, and fertilizer.

32. The engineered yeast cell of any one of claims 1-31, wherein the heterologous nucleic acid comprises a signal peptide.

33. The engineered yeast cell of claim 32, wherein the signal peptide is as provided for in WO2022 / 192675.

34. The engineered yeast cell of claim 32, wherein the signal peptide comprises a pre-protein signal peptide, a pro-protein signal peptide, or a combination thereof.

35. The engineered yeast cell of claim 33, wherein the pre-protein signal peptide is selected from: MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33), MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61), MKFKLTLLAAILAYANTVLVAS (SEQ ID NO:62), MKFKLTLLAALSALAALALAAP (SEQ ID NO:63), or MKFKLTIFAALLALAALALAAP (SEQ ID NO:64).

36. The engineered yeast cell of claim 33 or 34, wherein the pro-protein signal peptide is selected from: EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), or DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36).

37. The engineered yeast cell of any one of claims 33-35, wherein the signal peptide comprises: MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKLTLLAALLALAALVLAAS (SEQ ID NO: 31) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to 97 IPTS / 200107384.1Attorney Matter No. TNZ-017WO EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKLSSILLLLALLALVLAAS (SEQ ID NO: 32) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKLLSLLALLLLLASLVLAAS (SEQ ID NO: 33) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKSALFAALLALAALVLAAP (SEQ ID NO: 61) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKFKLTLLAAILAYANTVLVAS (SEQ ID NO: 62) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTLLAAILAYANTVLVAS (SEQ ID NO: 62) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKLTLLAAILAYANTVLVAS (SEQ ID NO: 62) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKFKLTLLAALSALAALALAAP (SEQ ID NO: 63) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTLLAALSALAALALAAP (SEQ ID NO: 63) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKLTLLAALSALAALALAAP (SEQ ID NO: 63) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36), MKFKLTIFAALLALAALALAAP (SEQ ID NO: 64) fused to EPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRHKRDV (SEQ ID NO: 34), MKFKLTIFAALLALAALALAAP (SEQ ID NO: 64) fused to STLTPSVVFIGGGLTEETTFGIRHKRDV (SEQ ID NO: 35), MKFKLTIFAALLALAALALAAP (SEQ ID NO: 64) fused to DPWSTTTSIYSLGGTTSYVSEFGLSISDETVTEMKSRHKRDV (SEQ ID NO: 36). 98 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 38. The engineered yeast cell of claim 32 or 34, wherein the signal peptide comprises the sequence of MKFKLTLLAALLALAALVLAASEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASR HKRDV (SEQ ID NO: 37).

39. The engineered yeast cell of claim 32 or 34, wherein the signal peptide comprises the sequence of MKFKSALFAALLALAALVLAAPEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASR HKRDV (SEQ ID NO: 65).

40. The engineered yeast cell of claim 32 or 34, wherein the signal peptide comprises the sequence of MKFKLTLLAAILAYANTVLVASEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRH KRDV (SEQ ID NO: 66).

41. The engineered yeast cell of claim 32 or 34, wherein the signal peptide comprises the sequence of MKFKLTLLAALSALAALALAAPEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASR HKRDV (SEQ ID NO: 67).

42. The engineered yeast cell of claim 32 or 34, wherein the signal peptide comprises the sequence of MKFKLTIFAALLALAALALAAPEPWSTLTVTRSTYDEITDTDYNSTGIAVNPYTVSASRH KRDV (SEQ ID NO: 68).

43. The engineered yeast cell of any one of claims 1-58, wherein the heterologous nucleic acid comprises a homology arm at the 5’ end and a homology arm at the 3’ end of the heterologous nucleic acid.

44. The engineered yeast cell of claim 43, wherein the homology arm at the 5’ end and the homology arm at the 3’ end of the heterologous nucleic acid comprise a sequence complementary to the 5’ insertion site and 3’ insertion stie, respectively.

45. The engineered yeast cell of claim 43 or 44, wherein the homology arm at the 5’ end comprises a complementary sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-3 and 7-18.

46. The engineered yeast cell of claim 43 or 44, wherein the homology arm at the 3’ end comprises a sequence with at least 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4-6 and 19-30.

47. The engineered yeast cell of any one of claims 1-46, wherein the heterologous nucleic acid comprises a nucleic acid sequence encoding an anchoring protein.

48. The engineered yeast cell of claim 47, wherein the anchoring protein is Sed1 or CWP2. 99 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 49. The engineered yeast cell of claim 47 or 48, wherein the anchoring protein is Sed1 and comprises a sequence of SEQ ID NO:

69.

50. The engineered yeast cell of claim 47 or 48, wherein the anchoring protein is CWP2 and comprises a sequence of SEQ ID NO:

70.

51. The engineered yeast cell of any one of claims 1-50, wherein the heterologous nucleic acid comprises a constitutive or inducible promoter.

52. The engineered yeast cell of claim 51, wherein the constitutive or inducible promoter is a TEF1 promoter or a TEF1 U3 promoter.

53. The engineered yeast cell of any one of claims 1-52, wherein the heterologous nucleic acid comprises one or two terminator sequences.

54. The engineered yeast cell of claim 53, wherein the one or two terminator sequences are a CYC1 terminator or a ADH1 terminator.

55. The engineered yeast cell of any one of claims 17-54, wherein the heterologous nucleic acid is integrated at 14 safe harbor sites and comprises a nucleic acid sequence that encodes invertase.

56. The engineered yeast cell of claim 55, wherein the heterologous nucleic acid comprises the following elements operably linked from 5’ to 3’: a CYC1 terminator sequence, a TEF1 promoter sequence, a Sb propeptide sequence, a sequence that encodes invertase, and a ADH1 terminator sequence.

57. The engineered yeast cell of any one of claims 1-56, further comprising a nuclease capable of generating a break in the 5’ integration site or 3’ integration site.

58. The engineered yeast cell of claim 57, wherein the nuclease is a nuclease selected from the group comprising TALEN, ZFN, a Cas protein, and HO endonuclease.

59. The engineered yeast cell of claim 58, wherein the nuclease is a Cas protein.

60. The engineered yeast cell of claim 58 or 59, wherein the nuclease is Cas9.

61. A method of making the engineered yeast cell of any one of claims 1-60 comprising targeted insertion of a heterologous nucleic acid into one or more safe harbor sites.

62. The method of claim 61, where the one or more safe harbor sites each comprise a 5’ integration site and a 3’ integration site.

63. The method of claim 61 or 62, comprising a nuclease capable of generating a break in the 5’ integration site or 3’ integration site.

64. The method of any one of claims 61-63, wherein the nuclease is a nuclease selected from the group comprising TALEN, ZFN, a Cas protein, and HO endonuclease.

65. The method of any one of claims 61-64, wherein the nuclease is a Cas protein.

66. The method of any one of claims 61-65, wherein the nuclease is Cas9. 100 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 67. A method for treating a disease or a condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the engineered yeast cell of any one of claims 1- 60.

68. The method of claim 67, wherein the disease or condition is selected from an infection, an autoimmune disease, primary (congenital) enzymatic deficiency, enzymatic deficiencies secondary to functional gut disorders, diabetes, obesity, a metabolic disorder, intestinal bacterial overgrowth, enteric infection, bacterial vaginosis, inflammatory bowel disease, irritable bowel syndrome, small bowel syndrome, Celiac disease, gluten intolerance, colitis, peptic ulcer, or another GI condition or disorder.

69. The method of claim 67 or 68, wherein the disease or condition is an enzyme deficiency and the payload protein is an enzyme.

70. The method of claim 67 or 68, wherein the disease or condition is congenital sucrase- isomaltase deficiency and the payload protein is one or both of invertase and isomaltase.

71. The method of claim 67 or 68, wherein the disease or condition is one or both of sucrose and isomaltase intolerance secondary to a functional gut disorder and the payload protein is one or both of invertase and isomaltase.

72. The method of claim 67 or 68, wherein the disease or condition is one or more of gluten intolerance, refractory sprue, or Celiac disease and the payload protein is one or more of An- PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide.

73. The method of claim 67 or 68, wherein the disease or condition is pancreatitis or exocrine pancreatic insufficiency and the payload protein is selected from one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin.

74. The method of claim 67 or 68, wherein the disease or condition is enteropeptidase deficiency or enterokinase deficiency and the payload protein is one or all of enteropeptidase, proenteropeptidase, and enterokinase.

75. The method of claim 67 or 68, wherein the disease or condition is small intestinal bacterial overgrowth, inflammatory bowel disease, irritable bowel syndrome, C. difficile infection, cystic fibrosis, necrotizing enterocolitis, and diabetes, and the payload protein is intestinal alkaline phosphatase.

76. The method of claim 67 or 68, wherein the disease or condition is caused by, or present with, endotoxemia, and the payload protein is human alkaline phosphatase, yeast alkaline phosphatase, bovine alkaline phosphatase, Cobetia amphilecti alkaline phosphatase, Paernibacillus lentus alkaline phosphatase, human lactoferrin (LF-22), human cathelicidin (LL- 37), human β-defensin-3 (HβD-3), human α-defensin 5 (HD5), human acyloxyacyl hydrolase (AOAH), Limulus polyphemus anti-lipopolysaccharide factor (ALF-L), tachypleus plasma lectin 101 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 2 (TPL2), human bactericidal permeability-increasing protein (BPI), and Crassostrea gigas bactericidal permeability-increasing protein (CgBPI).

77. The method of claim 76, wherein the disease or condition is aging, anxiety, autism, Alzheimer’s disease, Amyotrophic lateral sclerosis, atherosclerosis, chronic fatigue syndrome, chronic heart disease, cirrhosis, depression, diabetes, exercise-induced enteropathy, HIV enteropathy, metabolic endotoxemia, NSAID enteropathy, nonalcoholic fatty liver disease, inflammatory bowel disease, irritable bowel syndrome, obesity, Parkinson’s disease, sepsis, small intestinal bacterial overgrowth, rheumatoid arthritis, or thrombosis.

78. The method of claim 67 or 68, wherein the disease or condition is short bowel syndrome and the payload protein is IGF-1, GLP-2, or a synthetic derivative of GLP-2.

79. The method of claim 67 or 68, wherein the disease or condition is lactose sensitivity or lactose intolerance and the payload protein is lactase.

80. The method of claim 67 or 68, wherein the disease or condition is trehalose sensitivity or lactose intolerance and the payload protein is trehalase.

81. The method of claim 67 or 68, wherein the disease or condition is maltose sensitivity or lactose intolerance and the payload protein is maltase.

82. The method of claim 67 or 68, wherein the disease or condition is pernicious anemia and the payload protein is intrinsic factor.

83. The method of claim 67 or 68, wherein the disease or condition is bacterial overgrowth and the payload protein is lysozyme, nisin, a defensin, magainin, cateslytin, or any combination thereof.

84. The method of claim 67 or 68, wherein the condition is a bacterial infection caused by one or more of E. coli, C. difficile, vibrio cholera, Shigella, Salmonella, Cryptosporidium, or any combination thereof.

85. The method of claim 67 or 68, wherein the condition is a viral infection.

86. The method of claim 67 or 68, wherein the disease or condition is type 1 or type 2 diabetes mellitus and the payload protein is insulin, or an incretin.

87. The method of claim 67 or 68, wherein the administering is oral or topical.

88. The method of claim 67 or 68, wherein the disease or condition has an inflammatory component and the payload protein is IL-2, IL-10, IL-21, IL-22, TGFβ, an anti-TNFα antibody or fragment thereof, or any combination thereof.

89. The method of claim 67 or 68, wherein the disease or condition has an inflammatory component and the payload protein is IL-10.

90. The method of claim 67 or 68, wherein the disease or condition has an inflammatory component and the payload protein is IL-21. 102 IPTS / 200107384.1Attorney Matter No. TNZ-017WO 91. The method of claim 67 or 68, wherein the disease or condition has an inflammatory component and the payload protein is IL-22.

92. The method of claim 67 or 68, wherein the disease or condition has an inflammatory component and the payload protein is IL-2. 103 IPTS / 200107384.1

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