The term “allele” refers to a variation of a gene; the variations include but are not limited to variants and mutants, polymorphic loci, and single nucleotide polymorphic loci, frameshift, and splice mutations. An allele may occur naturally in a population, or it might arise during the lifetime of any particular individual of the population.

Thus, the terms “variant” and “mutant” when used in reference to a nucleotide sequence refer to a nucleic acid sequence that differs by one or more nucleotides from another, usually related, nucleotide acid sequence. A “variation” is a difference between two different nucleotide sequences; typically, one sequence is a reference sequence.

2. COMPOSITIONS AND METHODS

Embodiments of the present disclosure include an engineered yeast cell comprising at least one genetic modification that enhances protein expression and/or secretion of a target protein or polypeptide. In accordance with these embodiments, the present disclosure provides engineered yeast cells that include one or more genetic modifications in one or more genes that are involved in protein expression and/or secretion. These engineered yeast cells can be used as a platform to, for example, generate engineered live biotherapeutics (LBPs) to be administered to a subject to treat a disease or condition, and/or as a biomanufacturing platform to generate biologics for the treatment of a disease or condition. As would be recognized by one of ordinary skill in the art, the genetically modified yeast strains of the present disclosure can be used to express/secrete any desired protein or polypeptide. In some embodiments, the engineered yeast cell is from a probiotic strain. In some embodiments, the yeast cell is selected from the group consisting of S. boulardii, S. cerevisiae, P. pastoris, Y. lipolytica, C. albicans, K. marxianus, Debaryomyces hansenii, and Kluyveromyces lactis. In some embodiments, the yeast cell is from an S. boulardiiΔura3 strain (DD277).

In some embodiments, the engineered yeast cells of the present disclosure include at least one genetic modification in a gene involved in the protein secretion pathway. In some embodiments, the genetic medication results in the reduction of the activity and/or expression of the gene(s) involved in the protein secretion pathway. In some embodiments, the genetic modification is a gene knockout or a loss-of-function mutation. As described further herein, the genetic modification can be in any one of the following genes: APE1, YPS1, PRB1, PEP4, PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1. In accordance with these embodiments, expression of a target polypeptide or protein is increased compared to a yeast cell lacking at least one genetic modification in one or more of these genes (FIG. 3).

As would be recognized by one of ordinary skill in the art based on the present disclosure, the gene APE1 encodes Vacuolar aminopeptidase 1 (Uniprot No. P14904). The YPS1 gene encodes Aspartic proteinase 3 (Uniprot No. P32329). The PRB1 gene encodes Cerevisin (Uniprot No. P09232). The PEP4 gene encodes Vacuolar proteinase A (Uniprot No. A6ZW99). The PAH1 gene encodes Phosphatidic acid phosphohydrolase 1 (Uniprot No. P32567). The DER1 gene encodes Degradation in the endoplasmic reticulum protein 1 (Uniprot No. P38307). The HRD1 gene encodes ERAD-associated E3 ubiquitin-protein ligase (Uniprot No. Q08109). The OCH1 gene encodes Initiation-specific alpha-1,6-mannosyltransferase (Uniprot No. P31755). The MNN9 gene encodes Mannan polymerase complexes subunit MNN9 (Uniprot No. P39107). The VPS5 gene encodes Vacuolar protein sorting-associated protein vps5 (Uniprot No. Q9COU7). The VPS17 gene encodes Vacuolar protein sorting-associated protein 17 (Uniprot No. A6ZNX9). The TDA3 gene encodes Putative oxidoreductase TDA3 (Uniprot No. P38758). The GOS1 gene encodes Golgi SNAP receptor complex member 1 (Uniprot No. P38736). The ROX1 gene encodes Repressor ROX1 (Uniprot No. P25042).

A variety of genetic modifications (e.g., loss-of-function mutations) can be used in the various compositions and methods described herein. A genetic locus in a microorganism can produce one or more gene products. In some embodiments, the gene product is a nucleic acid, for example a ribosomal RNA. In some embodiments, the gene product is a polypeptide. A genetic modification affecting one or more genetic loci can cause a loss-of-function in the gene product, a gain-of-function in the gene product, and/or can cause the gene product to adopt a new function. In some embodiments, a loss-of-function mutation is provided that reduces, but does not eliminate activity of the gene product (this class of mutation can also be referred to herein a “hypomorphic” mutation or “partial loss-of-function” mutation). In some embodiments, a loss-of-function mutation substantially reduces gene product activity. As used herein, “substantially reduces” gene product activity and variations of this root term refer to at least a 50% reduction in gene product activity compared to wild-type, for example, a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 99.999% reduction. In some embodiments, a loss-of-function mutation eliminates gene product activity (this class of mutation can also be referred to herein as a “null” mutation). In some embodiments, a null mutation eliminates at least the gene-product-encoding sequence of a genetic locus. In some embodiments a null mutation eliminates the entire genetic locus. While a deletion of a genetic locus is a type of null mutation, null mutations can also encompass other sorts of genetic modifications. In some embodiments, a null mutation does not eliminate gene-product-encoding sequence, but prevents expression of the gene product (for example, by eliminating a promoter, a translation start sequence, or introducing an early stop codon). In some embodiments, a null mutation does not climinate gene-product-encoding sequence or expression of a gene product, but eliminates or substantially eliminates activity of the gene product (for example, by mutating one or more catalytic residues from a protein). This latter class of null mutation may also be referred to herein as a “phenotypic null,” or variations of this root term. In some embodiments, a loss-of-function mutation is a dominant negative mutation.

In addition to mutation of a genetic locus, other genetic modifications can also reduce or eliminate gene activity. By way of non-limiting example, antisense oligonucleotides can reduce or eliminate activity of the target gene. In some embodiments, a genetic modification for reducing gene expression comprises at least one antisense oligonucleotide. In some embodiments, an antisense oligonucleotide comprises an RNA complementary to at least a portion of a transcript of a target gene. In some embodiments, a yeast cell is genetically modified to express an antisense RNA directed to at least one transcript of the target gene. Additional exemplary genetic modifications that can be used to reduce gene activity in accordance with some embodiments herein include ribozymes, transcriptional repressors, inducible promoters, proteases directed to polypeptide encoded by a target gene, and the like.

In some embodiments, a mutation or genetic modification eliminates the activity of two or more gene products. In some embodiments, a mutation deletes an operon. In some embodiments, a mutation eliminates activity of one gene, and as a result, also eliminates activity of a second gene (for example, if the products of gene A and gene B function as a dimer, the elimination of either of gene A activity or gene B activity can also eliminate activity of the other gene).

A variety of techniques for making mutations are known to the skilled artisan. In some embodiments, a desired mutation is introduced via homologous recombination. A variety of vectors can be used for homologous mutation, for example phage or viral vectors, plasmid vectors, artificial chromosomes, and the like. In some embodiments, homologous sequences on a vector flank a genetic locus that can be used to identify homologous recombinants, for example an antibiotic resistance marker (for example, but not limited to kanamycin, chloramphenicol, or ampicillin resistance) or metabolic enzyme that permits an auxotroph to survive in a particular minimal medium. In some embodiments, mutations are introduced into a genome randomly, and mutant microorganisms having the desired mutations are selected.

In some embodiments, a host genome or portion thereof is synthesized, and introduced into a microorganism. In some embodiments an entire host genome having the desired genetic features is synthesized and inserted into a yeast cell.

In some embodiments, the at least one genetic modification comprises a genetic knockout of or a loss-of-function mutation in APE1. In some embodiments, the engineered yeast cells comprises a genetic knockout of or a loss-of-function mutation in APE1 and at least one other gene selected from the group consisting of YPS1, PRB1, PEP4, PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1. In some embodiments, the engineered yeast cell comprises a genetic knockout of or a loss-of-function mutation in APE1 and at least two other genes, at least three other genes, at least four other genes, or at least five other genes selected from the group consisting of YPS1, PRB1, PEP4, PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1.

In some embodiments, the at least one genetic modification comprises a genetic knockout of or a loss-of-function mutation in APE1. In some embodiments, the engineered yeast cells comprises a genetic knockout of or a loss-of-function mutation in YPS1 and at least one other gene selected from the group consisting of APE1, PRB1, PEP4, PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1. In some embodiments, the engineered yeast cell comprises a genetic knockout of or a loss-of-function mutation in YPS1 and at least two other genes, at least three other genes, at least four other genes, or at least five other genes selected from the group consisting of APE1, PRB1, PEP4, PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1.

In some embodiments, the at least one genetic modification comprises a genetic knockout of or a loss-of-function mutation in PRB1. In some embodiments, the engineered yeast cells comprises a genetic knockout of or a loss-of-function mutation in YPS1 and at least one other gene selected from the group consisting of APE1, YPS1, PEP4, PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1. In some embodiments, the engineered yeast cell comprises a genetic knockout of or a loss-of-function mutation in PRB1 and at least two other genes, at least three other genes, at least four other genes, or at least five other genes selected from the group consisting of APE1, YPS1, PEP4, PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1.

In some embodiments, the at least one genetic modification comprises a genetic knockout of or a loss-of-function mutation in PEP4. In some embodiments, the engineered yeast cells comprises a genetic knockout of or a loss-of-function mutation in YPS1 and at least one other gene selected from the group consisting of APE1, YPS1, PRB1, PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1. In some embodiments, the engineered yeast cell comprises a genetic knockout of or a loss-of-function mutation in PEP4 and at least two other genes, at least three other genes, at least four other genes, or at least five other genes selected from the group consisting of APE1, YPS1, PRB1, PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1.

In some embodiments, the engineered yeast cell of the present disclosure comprises at least one genetic modification comprising a genetic knockout of or a loss-of-function mutation in PRB1 and PEP4. In some embodiments, the engineered yeast cell comprises a genetic knockout of or a loss-of-function mutation in PRB1 and PEP4, and at least one other gene, at least two other genes, at least three other genes, at least four other genes, or at least five other genes selected from the group consisting of APE1, YPS1, PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1.

In some embodiments, the engineered yeast cell of the present disclosure comprises at least one genetic modification comprising a genetic knockout of or a loss-of-function mutation in YPS1, PRB1, PEP4, and APE1. In some embodiments, the engineered yeast cell comprises a genetic knockout of or a loss-of-function mutation in YPS1, PRB1, PEP4, and APE1, and at least one other gene, at least two other genes, at least three other genes, at least four other genes, or at least five other genes selected from the group consisting of PAH1, DER1, HRD1, OCH1, MNN9, VPS5, VPS17, TDA3, GOS1, and ROX1.

In accordance with these embodiments, the present disclosure provides an engineered yeast cell comprising one of the genetic modifications described above, as well as at least one exogenous polynucleotide that encodes a target polypeptide or protein. In some embodiments, at least a portion of the exogenous polynucleotide encodes a protein or polypeptide that is endogenous to the engineered yeast cell. In other embodiments, at least a portion of the exogenous polynucleotide encodes a protein or polypeptide that is exogenous to the engineered yeast cell. In some embodiments, the exogenous polynucleotide encodes more than one protein or polypeptide that can be endogenous and/or exogenous to the engineered yeast cell.

In some embodiments, the exogenous polynucleotide comprises other features that facilitate protein expression and/or secretion in the engineered yeast cell. For example, the exogenous polypeptide can include a promoter upstream of the target polypeptide or protein in order to facilitate its expression. In some embodiments, the exogenous polynucleotide can include a secretion signal upstream of the target polypeptide or protein in order to facilitate its secretion from the engineered yeast cell. Although any suitable promoter or secretion signal can be used, as would be recognized by one of ordinary skill in the art, in some embodiments, the secretion signal comprises an alpha mating factor secretion signal. In some embodiments, the target polypeptide or protein is expressed and secreted from the engineered yeast cell. In some embodiments, the target polypeptide or protein is expressed on the surface of the engineered yeast cell.

In some embodiments, the engineered yeast cells of the present disclosure comprising at least one of the genetic modifications described herein exhibit increased expression, secretion, and/or cell surface display of the target polypeptide or protein. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 1.1-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 1.2-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 1.3-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 1.4-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 1.5-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 1.6-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 1.7-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 1.8-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 1.9-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 2.0-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 3.0-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 4.0-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 5.0-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 6.0-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 7.0-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 8.0-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 9.0-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 10.0-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 15.0-fold compared to a yeast cell lacking the genetic modification. In some embodiments, expression, secretion, and/or cell surface display of the target polypeptide or protein is increased at least 20.0-fold compared to a yeast cell lacking the genetic modification.

Although any target protein or polypeptide can be expressed in the engineered yeast cells of the present disclosure, in some embodiments, the target polypeptide or protein is an anti-cancer agent, an immune regulator, an anti-pathogenic agent, and/or a metabolic regulator. In some embodiments, the target polypeptide or protein is at least one of a DARPin, a lectin, a monoclonal antibody, a F(ab′)₂fragment, a Fab′/Fab fragment, a diabody, a scFv, a nanobody, and/or an affibody.

The term “target protein” as used herein refers to a polypeptide or protein produced by recombinant technology in a host cell. More specifically, the protein may be a polypeptide that does not occur naturally in the host cell (i.e., a heterologous protein), or may be natural to the host cell (i.e., a protein homologous to the host cell), for example, by transformation with a self-replicating vector containing a nucleic acid sequence encoding a target protein, or by integration of one or more copies of the nucleic acid sequence encoding a target protein into the genome of a host cell, or by integration techniques, e.g., a promoter produced by recombinant modification of one or more regulatory sequences that regulate expression of the gene encoding the target protein. In some embodiments, the target protein is preferably an antibody or fragment thereof, enzyme and peptide, protein antibiotic, toxin fusion protein, carbohydrate-protein conjugate, structural protein, regulatory protein, vaccine and vaccine-like protein or particle. The target protein can also be a recombinant or heterologous protein selected from therapeutic proteins including process enzymes, growth factors, hormones and cytokines or metabolites of target protein. An exemplary target protein is an antigen-binding molecule such as an antibody or fragment thereof. Among certain target proteins are monoclonal antibodies (mAb), immunoglobulins (Ig) or immunoglobulin class G (IgG), heavy chain antibodies (HcAb′), or fragments thereof, such as fragment-antigen binding (Fab), Fd, Single-chain variable fragments (scFv) or engineered variants thereof, such as Fv dimer (diabody), Fv trimer (tribody), Fv tetramer or minibody, and VH or VHH or V-NAR Antibodies such as single-domain antibodies.

In accordance with the above embodiments, the present disclosure also includes a composition comprising any of the engineered yeast cells described herein. In some embodiments, the composition is formulated as a food product or a medicament. In some embodiments, the composition is lyophilized. In some embodiments, the composition is in wet form. In some embodiments, the composition is in frozen form. As would be recognized by one of ordinary skill in the art based on the present disclosure, yeast cells can be formulated as a lyophilized composition (e.g., including a cryoprotectant) and can be readily reconstituted, which is an advantage over many other microorganisms (e.g., bacteria and fungi). In this aspect, the compositions of the present disclosure are particularly suited for lyophilization and formulation as an LBP and/or therapeutic composition for administration to a subject.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present disclosure include molecular, biochemical, microbiological and recombinant DNA techniques that are well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. Such techniques are thoroughly explained in the literature and are generally performed according to methods available to those of skill in the art. For example, the phrase “nucleic acid” or “polynucleotide sequence” refers to a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids may also include modified nucleotides that permit correct read-through by a polymerase and do not alter expression of a polypeptide encoded by that nucleic acid. A “coding sequence” or “coding region” refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed. The phrase “nucleic acid sequence encoding” refers to a nucleic acid which directs the expression of a specific protein or polypeptide. The nucleic acid sequences described in the present disclosure include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences. It should be understood that the sequences include the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

The terms “isolated,” “purified,” or “biologically pure” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using molecular biology and analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid of the present disclosure is separated from open reading frames that flank the desired gene and encode proteins other than the desired protein. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of an RNA and/or polypeptide, respectively. The expression cassette may include a nucleic acid comprising a promoter sequence, with or without a sequence containing mRNA polyadenylation signals, and one or more restriction enzyme sites located downstream from the promoter allowing insertion of heterologous gene sequences. The expression cassette is capable of directing the expression of a heterologous protein when the gene encoding the heterologous protein is operably linked to the promoter by insertion into one of the restriction sites. The recombinant expression cassette allows expression of the heterologous protein in a host cell when the expression cassette containing the heterologous protein is introduced into the host cell. Expression cassettes can be derived from a variety of sources depending on the host cell to be used for expression. For example, an expression cassette can contain components derived from a viral, bacterial, insect, plant, or mammalian source. In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or sense suppression) the inserted polynucleotide sequence need not be identical and can be “substantially identical” to a sequence of the gene from which it was derived. For example, some yeasts belong to the CUG clade which have an alternative codon usage and the polynucleotide must be altered to code for the correct amino acid and ensure that lys/ser are properly incorporated.

The term “recombinant cell” (or simply “host cell”) refers to a cell into which a recombinant expression vector containing the constructs described herein has been introduced. It should be understood that the term “host cell” is intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Methods for introducing polynucleotide sequences into various types of cells are well known in the art. Provided are host cells or progeny of host cells transformed with the recombinant expression cassettes and constructs of the present disclosure.

The terms “promoter,” “promoter region,” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5′ or 3′ l side of the coding region, or within the coding region, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. The typical 5′ promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

As used herein, a polynucleotide is “operably linked,” “operably connected,” or “operably inserted” when it is placed into a functional relationship with a second polynucleotide sequence. For instance, a promoter is operably linked to a coding sequence if the promoter is connected to the coding sequence such that it may affect transcription of the coding sequence. This same definition is sometimes applied to the arrangement of other transcription control elements (e.g., enhancers, terminators) in an expression cassette. Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. Promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally regulated, and chemically regulated promoters.

The term “nucleic acid construct” or “DNA construct” is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into an expression cassette for transforming a cell or for translating a protein in a cell-free system or for use in homologous recombination. Such a nucleic acid construct may contain a coding sequence for a gene product of interest, and optionally a selectable marker gene and/or a reporter gene. The term “selectable marker gene” refers to a gene encoding a product that, when expressed, confers a selectable phenotype, such as antibiotic resistance, on a transformed cell. The term “reporter gene” refers to a gene that encodes a product which is easily detectable by standard methods, either directly or indirectly. Reporter genes include, but are not limited to luciferases, ß-glucuronidase (GUS), fluorescent proteins such as green fluorescent protein (GFP), dsRed, mCherry and others available to those skilled in the art. Selectable markers include but are not limited to markers that confer resistance to an antibiotic such as kanamycin and hygromycin.

A “heterologous” region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature. When the heterologous region encodes a gene, the gene will usually be flanked by DNA that does not flank the genetic DNA in the genome of the source organism. In another example, a heterologous region is a construct where the coding sequence itself is not found in nature. The term “DNA construct” is also used to refer to a heterologous region, particularly one constructed for use in transformation of a cell.

The term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, where additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome, such as some viral vectors or transposons. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked or connected. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

A “nucleic acid probe” or “oligonucleotide” is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural bases (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. For example, probes may be peptide nucleic acids (PNAs) in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence (sequence fragment).

Embodiments of the present disclosure also include a method of treating and/or preventing a disease or condition in a subject by administering any of the engineered yeast cells or compositions comprising the engineered yeast cells described herein. In accordance with these embodiments, the methods include administering any of the compositions described herein to the subject. In some embodiments, the composition is administered orally, rectally, parenterally, intramuscularly, intraperitoneally, intravenously, intracerebroventricularly, intracisternally, subcutaneously, via injection or infusion, via inhalation, spray, nasal, vaginal, rectal, sublingual, or topical administration. In some embodiments, the composition is administered orally or rectally.

In some embodiments, the engineered yeast cells are present in the composition at a dose from about 1×10⁵cells/kg body weight to about 1×10¹⁵cells/kg body weight of the subject. In some embodiments, the engineered yeast cells are present in the composition at a dose from about 1×10⁵cells/kg body weight to about 1×10¹⁴cells/kg body weight of the subject. In some embodiments, the engineered yeast cells are present in the composition at a dose from about 1×10⁵cells/kg body weight to about 1×10